HK1198661B - Nucleic acid encoding reactions - Google Patents

Description

Translated fromChinese

核酸编码反应Nucleic acid encoding reaction

相关申请的交叉参考CROSS-REFERENCE TO RELATED APPLICATIONS

本申请要求2011年5月20日提交的美国临时申请号61/519,348的权益，其通过引用全文并入本文。This application claims the benefit of U.S. Provisional Application No. 61/519,348, filed May 20, 2011, which is incorporated herein by reference in its entirety.

发明领域Field of the Invention

本发明一般涉及掺入核酸序列到靶核酸中，例如，加入一个或多个衔接子(adaptor)和/或核苷酸标签和/或条形码核苷酸序列到靶核苷酸序列。本文描述的方法可用于，例如，用于检测和/或测序特定靶核酸的高通量检验领域中。The present invention generally relates to incorporating nucleic acid sequences into target nucleic acids, for example, adding one or more adapters and/or nucleotide tags and/or barcode nucleotide sequences to a target nucleotide sequence. The methods described herein can be used, for example, in the field of high-throughput assays for detecting and/or sequencing specific target nucleic acids.

发明背景Background of the Invention

在样品中检出特定核酸序列的能力已经导致诊断和预测医学、环境、食品和农业监测，分子生物学研究，以及多种其他领域中的多种新方法。对于许多应用，期望同时检测和/或分析多个样品中的许多靶核酸，所述多个样品例如，群体中的多个单独细胞。The ability to detect specific nucleic acid sequences in a sample has led to a variety of new methods in diagnostic and prognostic medicine, environmental, food and agricultural monitoring, molecular biology research, and a variety of other fields. For many applications, it is desirable to simultaneously detect and/or analyze many target nucleic acids in multiple samples, such as multiple individual cells in a population.

发明概述SUMMARY OF THE INVENTION

在某些实施方案中，本发明提供向包含粘末端的多个靶核酸的每个末端加入衔接子分子的方法。该方法包括(entail)退火衔接子分子到双链靶核酸分子的粘末端以产生退火的衔接子-靶核酸分子，其中衔接子分子是：In certain embodiments, the present invention provides a method for adding an adapter molecule to each end of a plurality of target nucleic acids comprising cohesive ends. The method entails annealing an adapter molecule to the cohesive ends of a double-stranded target nucleic acid molecule to produce annealed adapter-target nucleic acid molecules, wherein the adapter molecule is:

(i)发夹结构，所述发夹结构各自包含：(i) hairpin structures, each of the hairpin structures comprising:

衔接子核苷酸序列，其连接于Adaptor nucleotide sequence, which is connected to

核苷酸接头(linker)，其连接于Nucleotide linker, which is connected to

核苷酸序列，其能够退火到衔接子核苷酸序列并连接于nucleotide sequence that is capable of annealing to an adapter nucleotide sequence and ligating to

简并尾序列；或Degenerate tail sequence; or

(ii)双链或单链分子，所述双链或单链分子在每条链上各自包含：(ii) a double-stranded or single-stranded molecule comprising on each strand:

第一衔接子核苷酸序列，其连接于A first adaptor nucleotide sequence is ligated to

核苷酸接头，其连接于Nucleotide linker, which is connected to

第二衔接子核苷酸序列；和a second adaptor nucleotide sequence; and

简并尾序列，其中双链分子各自包含两个简并尾序列作为粘末端。在退火后，该方法包括填充所得的退火的衔接子-靶核酸分子中的任何缺口，和连接退火的衔接子-靶核酸分子中任何邻近的核苷酸序列以产生衔接子修饰的靶核酸分子。在相关的实施方案中，本发明提供多个衔接子分子，其中衔接子分子是以上(i)的发夹结构或以上(ii)的双链或单链分子。还涵盖了试剂盒，在多个实施方案中，该试剂盒可包含多个衔接子分子联合DNA酶、核酸外切酶、核酸内切酶、聚合酶、连接酶、或其任何组合。Degenerate tail sequence, wherein each double-stranded molecule comprises two degenerate tail sequences as sticky ends. After annealing, the method includes filling any gaps in the annealed adapter-target nucleic acid molecule obtained, and connecting any adjacent nucleotide sequences in the annealed adapter-target nucleic acid molecule to produce an adapter-modified target nucleic acid molecule. In a related embodiment, the present invention provides a plurality of adapter molecules, wherein the adapter molecule is a hairpin structure of (i) above or a double-stranded or single-stranded molecule of (ii) above. Also contemplated are kits, which, in multiple embodiments, may comprise a plurality of adapter molecules in combination with DNA enzymes, exonucleases, endonucleases, polymerases, ligases, or any combination thereof.

在其他实施方案中，本发明提供用核苷酸序列将多个靶核酸加标签的方法。该方法包括为每个靶核酸准备第一反应混合物，第一反应混合物包含内侧引物对和外侧引物对，其中：In other embodiments, the present invention provides a method for tagging a plurality of target nucleic acids with nucleotide sequences. The method comprises preparing a first reaction mixture for each target nucleic acid, the first reaction mixture comprising an inner primer pair and an outer primer pair, wherein:

(i)内侧引物包含：(i) The inner primer comprises:

正向、内侧引物，包含第一核苷酸标签、第一条形码核苷酸序列和靶特异性部分；和a forward, inner primer comprising a first nucleotide tag, a first barcode nucleotide sequence, and a target-specific portion; and

反向、内侧引物，包含靶特异性部分、第一条形码核苷酸序列和第二核苷酸标签；和a reverse, inner primer comprising a target-specific portion, a first barcode nucleotide sequence, and a second nucleotide tag; and

(ii)外侧引物包含：(ii) Outer primers comprising:

正向、外侧引物，包含第二条形码核苷酸序列和第一核苷酸标签特异性部分；和a forward, outer primer comprising a second barcode nucleotide sequence and a first nucleotide tag-specific portion; and

反向、外侧引物，包含第二核苷酸标签特异性部分和第二条形码核苷酸序列，其中外侧引物是比内侧引物过量的。对每个第一反应混合物进行反应以产生多个加标签的靶核苷酸序列，各自包含5'-第二条形码核苷酸序列-第一核苷酸标签序列-第一条形码核苷酸序列-靶核苷酸序列-第一条形码核苷酸序列-第二核苷酸标签序列-第二条形码核苷酸序列-3'。在相关的实施方案中，本发明提供试剂盒，该试剂盒包含聚合酶联合以上(i)的内侧引物和以上(ii)的外侧引物，其中外侧引物是比内侧引物过量的。A reverse, outer primer comprises a second nucleotide tag-specific portion and a second barcode nucleotide sequence, wherein the outer primer is present in excess relative to the inner primer. Each first reaction mixture is reacted to produce a plurality of tagged target nucleotide sequences, each comprising 5′-second barcode nucleotide sequence-first nucleotide tag sequence-first barcode nucleotide sequence-target nucleotide sequence-first barcode nucleotide sequence-second nucleotide tag sequence-second barcode nucleotide sequence-3′. In a related embodiment, the present invention provides a kit comprising a polymerase in combination with the inner primer of (i) above and the outer primer of (ii) above, wherein the outer primer is present in excess relative to the inner primer.

在某些实施方案中，本发明提供用核苷酸序列将多个靶核酸加标签的方法。该方法包括为每个靶核酸准备第一反应混合物，第一反应混合物包含内侧引物对、填充引物(stuffer primer)对和外侧引物对，其中：In certain embodiments, the present invention provides a method for tagging a plurality of target nucleic acids with nucleotide sequences. The method comprises preparing a first reaction mixture for each target nucleic acid, the first reaction mixture comprising an inner primer pair, a stuffer primer pair, and an outer primer pair, wherein:

(i)内侧引物包含：(i) The inner primer comprises:

正向、内侧引物，包含第一核苷酸标签和靶特异性部分；和a forward, inner primer comprising a first nucleotide tag and a target-specific portion; and

反向、内侧引物，包含靶特异性部分和第二核苷酸标签；a reverse, inner primer containing a target-specific portion and a second nucleotide tag;

(ii)填充引物包含：(ii) the filler primer comprises:

正向、填充引物，包含第三核苷酸标签、第一条形码核苷酸序列和第一核苷酸标签特异性部分；和a forward, filler primer comprising a third nucleotide tag, a first barcode nucleotide sequence, and a portion specific for the first nucleotide tag; and

反向、填充引物，包含第二核苷酸标签特异性部分、第一条形码核苷酸序列、第四核苷酸标签；和a reverse, filler primer comprising a second nucleotide tag-specific portion, a first barcode nucleotide sequence, a fourth nucleotide tag; and

(iii)外侧引物包含：(iii) Outer primers comprising:

正向、外侧引物，包含第二条形码核苷酸序列和第三核苷酸标签特异性部分；和a forward, outer primer comprising a second barcode nucleotide sequence and a third nucleotide tag-specific portion; and

反向、外侧引物，包含第四核苷酸标签特异性部分和第二条形码核苷酸序列，其中外侧引物是比填充引物过量的，填充引物是比内侧引物过量的。对每个第一反应混合物进行反应以产生多个加标签的靶核苷酸序列，各自包含5'-第二条形码核苷酸序列-第三核苷酸标签序列-第一条形码核苷酸序列-第一核苷酸标签序列-靶核苷酸序列-第二核苷酸标签序列-第一条形码核苷酸序列-第四核苷酸标签序列-第二条形码核苷酸序列-3'。在相关的实施方案中，本发明提供试剂盒，该试剂盒包含聚合酶联合以上(i)的内侧引物、以上(ii)的填充引物、和以上(iii)的外侧引物，其中外侧引物是比填充引物过量的，填充引物是比内侧引物过量的。A reverse, outer primer comprises a fourth nucleotide tag-specific portion and a second barcode nucleotide sequence, wherein the outer primer is present in excess of the filler primer, which is present in excess of the inner primer. Each first reaction mixture is reacted to produce a plurality of tagged target nucleotide sequences, each comprising 5′-second barcode nucleotide sequence-third nucleotide tag sequence-first barcode nucleotide sequence-first nucleotide tag sequence-target nucleotide sequence-second nucleotide tag sequence-first barcode nucleotide sequence-fourth nucleotide tag sequence-second barcode nucleotide sequence-3′. In a related embodiment, the present invention provides a kit comprising a polymerase in combination with the inner primer of (i), the filler primer of (ii), and the outer primer of (iii), wherein the outer primer is present in excess of the filler primer, which is present in excess of the inner primer.

在具体实施方案中，本发明提供将多个靶核苷酸序列组合加标签(combinatorialtagging)的方法。该方法采用来源于靶核酸的多个加标签的靶核苷酸序列(a pluralityof tagged target nucleotide sequences)，每个加标签的靶核苷酸序列包含核酸内切酶位点和第一条形码核苷酸序列，其中多个加标签的靶核苷酸序列(tagged targetnucleotide sequences in the plurality)包含相同的核酸内切酶位点、但N个不同的第一条形码核苷酸序列，其中N是大于1的整数。该方法包括用对核酸内切酶位点特异性的核酸内切酶切割多个加标签的靶核苷酸序列以产生多个具有粘末端的(sticky-ended)、加标签的靶核苷酸序列。该方法还包括在第一反应混合物中连接包含第二条形码核苷酸序列和互补粘末端的多个衔接子于多个具有粘末端的、加标签的靶核苷酸序列，其中多个衔接子包含M个不同的第二条形码核苷酸序列，其中M是大于1的整数。这一连接产生多个组合加标签的靶核苷酸序列，各自包含第一和第二条形码核苷酸序列，其中多个包括N x M个不同的第一和第二条形码的组合。在相关的实施方案中，本发明提供多个衔接子，包含：In a specific embodiment, the present invention provides a method for combinatorial tagging of multiple target nucleotide sequences. The method uses a plurality of tagged target nucleotide sequences derived from a target nucleic acid, each tagged target nucleotide sequence comprising an endonuclease site and a first barcode nucleotide sequence, wherein the plurality of tagged target nucleotide sequences comprises the same endonuclease site but N different first barcode nucleotide sequences, where N is an integer greater than 1. The method comprises cleaving the plurality of tagged target nucleotide sequences with an endonuclease specific for the endonuclease site to produce a plurality of sticky-ended tagged target nucleotide sequences. The method further comprises ligating a plurality of adaptors comprising second barcode nucleotide sequences and complementary sticky ends to the plurality of sticky-ended tagged target nucleotide sequences in a first reaction mixture, wherein the plurality of adaptors comprises M different second barcode nucleotide sequences, where M is an integer greater than 1. This ligation produces a plurality of combinatorially tagged target nucleotide sequences, each comprising a first and a second barcode nucleotide sequence, wherein the plurality comprises N x M different combinations of first and second barcodes. In a related embodiment, the invention provides a plurality of adaptors comprising:

多个第一衔接子，各自包含相同的核酸内切酶位点、N个不同的条形码核苷酸序列、第一引物结合位点和粘末端，其中N是大于1的整数；a plurality of first adaptors, each comprising an identical endonuclease site, N different barcode nucleotide sequences, a first primer binding site, and sticky ends, wherein N is an integer greater than 1;

第二衔接子，包含第二引物结合位点和粘末端；和a second adaptor comprising a second primer binding site and sticky ends; and

多个第三衔接子，包含第二条形码核苷酸序列和与在所述核酸内切酶位点切割所述第一衔接子时产生的那些互补的粘末端，其中多个第三衔接子包含M个不同的第二条形码核苷酸序列，其中M是大于1的整数。还涵盖的是试剂盒，该试剂盒包含多个第一衔接子、第二衔接子、和多个第三衔接子、联合对第一衔接子中的核酸内切酶位点特异性的核酸内切酶和/或连接酶。a plurality of third adaptors comprising a second barcode nucleotide sequence and sticky ends complementary to those produced upon cleavage of the first adaptor at the endonuclease site, wherein the plurality of third adaptors comprises M different second barcode nucleotide sequences, where M is an integer greater than 1. Also contemplated are kits comprising a plurality of first adaptors, a second adaptor, and a plurality of third adaptors in combination with an endonuclease and/or a ligase specific for the endonuclease site in the first adaptor.

在其他实施方案中，本发明提供用于将多个靶核苷酸序列组合加标签的方法，其中该方法包括退火多个条形码引物到来源于靶核酸的多个加标签的靶核苷酸序列。每个加标签的靶核苷酸序列包含在一个末端的核苷酸标签和第一条形码核苷酸序列，其中多个加标签的靶核苷酸序列包含相同的核苷酸标签、但N个不同的第一条形码核苷酸序列，其中N是大于1的整数。每个条形码引物包含：In other embodiments, the present invention provides a method for combinatorially tagging a plurality of target nucleotide sequences, wherein the method comprises annealing a plurality of barcode primers to a plurality of tagged target nucleotide sequences derived from a target nucleic acid. Each tagged target nucleotide sequence comprises a nucleotide tag at one end and a first barcode nucleotide sequence, wherein the plurality of tagged target nucleotide sequences comprise the same nucleotide tag but N different first barcode nucleotide sequences, where N is an integer greater than 1. Each barcode primer comprises:

第一标签特异性部分，连接于；a first tag-specific portion, attached to;

第二条形码核苷酸序列，连接于；a second barcode nucleotide sequence, linked to;

第二标签特异性部分，其中多个条形码引物各自包含相同的第一和第二标签特异性部分、但M个不同的第二条形码核苷酸序列，其中M是大于1的整数。该方法还包括在第一反应混合物中扩增加标签的靶核苷酸序列以产生多个组合加标签的靶核苷酸序列，各自包含第一和第二条形码核苷酸序列，其中多个包含NxM个不同的第一和第二条形码组合在相关的实施方案中，本发明提供试剂盒，该试剂盒包含一个或多个核苷酸标签连同以上的多个条形码引物，核苷酸标签可用于产生加标签的靶核苷酸序列。and a second tag-specific portion, wherein the plurality of barcode primers each comprise the same first and second tag-specific portion but M different second barcode nucleotide sequences, where M is an integer greater than 1. The method further comprises amplifying the tagged target nucleotide sequence in the first reaction mixture to generate a plurality of combinatorially tagged target nucleotide sequences, each comprising the first and second barcode nucleotide sequence, wherein the plurality comprises N x M different first and second barcode combinations. In related embodiments, the present invention provides a kit comprising one or more nucleotide tags together with the plurality of barcode primers above, the nucleotide tags being useful for generating tagged target nucleotide sequences.

在某些实施方案中，本发明提供用于检测多个靶核酸的检验方法，该方法包括准备将在检验前被汇集的M个第一反应混合物，其中M是大于1的整数。每个第一反应混合物包含：In certain embodiments, the present invention provides an assay for detecting a plurality of target nucleic acids, the method comprising preparing M first reaction mixtures to be pooled prior to assay, wherein M is an integer greater than 1. Each first reaction mixture comprises:

样品核酸；Sample nucleic acid;

第一、正向引物，包含靶特异性部分；First, the forward primer, which contains the target-specific portion;

第一、反向引物，包含靶特异性部分，其中第一、正向引物或第一、反向引物另外包含条形码核苷酸序列，且其中M个反应混合物的每一个中的每个条形码核苷酸序列是不同的。对每个第一反应混合物进行第一反应以产生多个条形码化的靶核苷酸序列，各自包含连接于条形码核苷酸序列的靶核苷酸序列。该方法还包括对于M个第一反应混合物的每一个，汇集所述条形码化的靶核苷酸序列以形成检验池。使用独特的第二引物对对检验池或其一个或多个等份进行第二反应，其中每个第二引物对分别包含：A first, reverse primer comprising a target-specific portion, wherein the first, forward primer or the first, reverse primer further comprises a barcode nucleotide sequence, and wherein each barcode nucleotide sequence in each of the M reaction mixtures is different. Each first reaction mixture is subjected to a first reaction to produce a plurality of barcoded target nucleotide sequences, each comprising a target nucleotide sequence linked to a barcode nucleotide sequence. The method further comprises, for each of the M first reaction mixtures, pooling the barcoded target nucleotide sequences to form a test pool. A second reaction is performed on the test pool or one or more aliquots thereof using a unique second primer pair, wherein each second primer pair comprises:

第二、正向或反向引物，退火到靶核苷酸序列；和Second, a forward or reverse primer, annealing to the target nucleotide sequence; and

第二、反向或正向引物，退火到条形码核苷酸序列。该方法然后包括对每个独特的、第二引物对，确定反应产物是否存在于检验池或其等份中，藉以反应产物的存在指示特定第一反应混合物中特定靶核酸的存在。A second, reverse or forward primer, anneals to the barcode nucleotide sequence. The method then includes determining, for each unique, second primer pair, whether a reaction product is present in the test pool or an aliquot thereof, whereby the presence of the reaction product indicates the presence of a specific target nucleic acid in the specific first reaction mixture.

在具体实施方案中，用于检测多个靶核酸的这一检验方法的变化形式包括准备将在检验前被汇集的M个第一反应混合物，其中M是大于1的整数，且每个第一反应混合物包含：In a specific embodiment, a variation of this assay for detecting a plurality of target nucleic acids comprises preparing M first reaction mixtures to be pooled prior to the assay, wherein M is an integer greater than 1, and each first reaction mixture comprises:

样品核酸；Sample nucleic acid;

第一、正向引物，包含靶特异性部分；First, the forward primer, which contains the target-specific portion;

第一、反向引物，包含靶特异性部分，其中第一、正向引物或第一、反向引物另外包含核苷酸标签；和a first, reverse primer comprising a target-specific portion, wherein the first, forward primer or the first, reverse primer additionally comprises a nucleotide tag; and

至少一个条形码引物，包含条形码核苷酸序列和核苷酸标签特异性部分，其中条形码引物是比第一、正向和/或第一、反向引物过量的，且其中M个反应混合物的每一个中的每个条形码核苷酸序列是不同的。对每个第一反应混合物进行第一反应以产生多个条形码化的靶核苷酸序列，各自包含靶核苷酸序列连接于核苷酸标签连接于条形码核苷酸序列(atarget nucleotide sequence linked to a nucleotide tag,which is linked to abarcode nucleotide sequence)。该方法还包括对于M个反应混合物的每一个，汇集条形码化的靶核苷酸序列以形成检验池。使用独特的第二引物对对检验池或其一个或多个等份进行第二反应，其中每个第二引物对分别包含：At least one barcode primer comprises a barcode nucleotide sequence and a nucleotide tag-specific portion, wherein the barcode primer is in excess of the first forward and/or first reverse primer, and wherein each barcode nucleotide sequence in each of the M reaction mixtures is different. Each first reaction mixture is subjected to a first reaction to produce a plurality of barcoded target nucleotide sequences, each comprising a target nucleotide sequence linked to a nucleotide tag, which is linked to a barcode nucleotide sequence. The method further comprises, for each of the M reaction mixtures, pooling the barcoded target nucleotide sequences to form a test pool. A second reaction is performed on the test pool or one or more aliquots thereof using a unique second primer pair, wherein each second primer pair comprises:

第二、正向或反向引物，其退火到靶核苷酸序列；和Second, a forward or reverse primer, which anneals to the target nucleotide sequence; and

第二、反向或正向引物，其退火到条形码核苷酸序列。该方法然后包括对每个独特的、第二引物对，确定反应产物是否存在于检验池或其等份中，藉以反应产物的存在指示特定第一反应混合物中特定靶核酸的存在。A second, reverse or forward primer is prepared that anneals to the barcode nucleotide sequence. The method then includes determining, for each unique, second primer pair, whether a reaction product is present in the test pool or an aliquot thereof, whereby the presence of the reaction product indicates the presence of a specific target nucleic acid in the specific first reaction mixture.

在某些实施方案中，本发明提供可用于扩增一个或多个靶核酸用于准备应用诸如双向核酸测序的方法和试剂盒。在一些实施方案中，本发明的方法包括另外进行双向DNA测序。In certain embodiments, the present invention provides methods and kits that can be used to amplify one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In some embodiments, the methods of the present invention include additionally performing bidirectional DNA sequencing.

在特定的双向实施方案中，这些方法包括扩增、加标签和条形码化多个样品中的多个靶核酸。核苷酸标签序列可包含可用于帮助扩增和/或DNA测序的引物结合位点。条形码核苷酸序列可编码关于扩增产物的信息，诸如从中得到该扩增产物的样品的身份。In certain bidirectional embodiments, these methods include amplifying, tagging, and barcoding multiple target nucleic acids in multiple samples. The nucleotide tag sequence can contain primer binding sites that can be used to facilitate amplification and/or DNA sequencing. The barcode nucleotide sequence can encode information about the amplification product, such as the identity of the sample from which the amplification product was obtained.

在某些双向实施方案中，扩增靶核酸的方法包括利用以下扩增靶核酸：In certain bidirectional embodiments, the method of amplifying a target nucleic acid comprises amplifying the target nucleic acid using:

内侧引物组，其中该组包含：An inner primer set, wherein the set comprises:

内侧、正向引物，包含靶特异性部分和第一引物结合位点；an inner, forward primer containing a target-specific portion and a first primer binding site;

内侧、反向引物，包含靶特异性部分和第二引物结合位点，其中第一和第二引物结合位点是不同的；an inner, reverse primer comprising a target-specific portion and a second primer binding site, wherein the first and second primer binding sites are different;

第一外侧引物组，其中该组包含：A first outer primer set, wherein the set comprises:

第一外侧、正向引物，包含对第一引物结合位点特异性的部分；和a first outer, forward primer comprising a portion specific for the first primer binding site; and

第一外侧、反向引物，包含条形码核苷酸序列和对第二引物结合位点特异性的部分；a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for the second primer binding site;

第二外侧引物组，其中该组包含：a second outer primer set, wherein the set comprises:

第二外侧、正向引物，包含条形码核苷酸序列和对第一引物结合位点特异性的部分；和a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and

第二外侧、反向引物，包含对第二引物结合位点特异性的部分。这一扩增产生两个靶扩增子，其中：The second outer, reverse primer contains a portion specific for the second primer binding site. This amplification produces two target amplicons, where:

第一靶扩增子包含5'-第一引物结合位点-靶核苷酸序列-第二引物结合位点-条形码核苷酸序列-3'；和The first target amplicon comprises 5'-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-3'; and

第二靶扩增子包含5'-条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-3'。在这些实施方案的变化形式中，每个靶扩增子中的条形码核苷酸序列是相同的，且每个靶扩增子包含仅一个条形码核苷酸序列。The second target amplicon comprises 5'-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-3'. In variations of these embodiments, the barcode nucleotide sequence in each target amplicon is identical, and each target amplicon comprises only one barcode nucleotide sequence.

在一些双向实施方案中，第一和第二引物结合位点是DNA测序引物的结合位点。外侧引物可任选地各自另外包含另外的核苷酸序列，其中：In some bidirectional embodiments, the first and second primer binding sites are binding sites for DNA sequencing primers. The outer primers may optionally each further comprise an additional nucleotide sequence, wherein:

第一外侧、正向引物包含第一另外的核苷酸序列，且第一外侧、反向引物包含第二另外的核苷酸序列；和The first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and

第二外侧、正向引物包含第二另外的核苷酸序列，且第二外侧、反向引物包含所述第一另外的核苷酸序列；且第一和第二另外的核苷酸序列是不同的。在这样的实施方案中，扩增产生两个靶扩增子，其中：The second outer, forward primer comprises a second additional nucleotide sequence, and the second outer, reverse primer comprises the first additional nucleotide sequence; and the first and second additional nucleotide sequences are different. In such an embodiment, amplification produces two target amplicons, wherein:

第一靶扩增子包含：5'-第一另外的核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-条形码核苷酸序列-第二另外的核苷酸序列-3'；和The first target amplicon comprises: 5′-first additional nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-second additional nucleotide sequence-3′; and

第二靶扩增子包含：5'-第二另外的核苷酸序列-条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一另外的核苷酸序列3'。在具体实施方案中，第一和/或第二另外的核苷酸序列包含引物结合位点。在示例性实施方案中，第一外侧引物组包含PE1-CS1和PE2-BC-CS2，且第二外侧引物组包含PE1-CS2和PE2-BC-CS1(表1，实施例9)。The second target amplicon comprises: 5'-second additional nucleotide sequence-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first additional nucleotide sequence 3'. In specific embodiments, the first and/or second additional nucleotide sequence comprises a primer binding site. In an exemplary embodiment, the first outer primer set comprises PE1-CS1 and PE2-BC-CS2, and the second outer primer set comprises PE1-CS2 and PE2-BC-CS1 (Table 1, Example 9).

在某些双向实施方案中，扩增在单个扩增反应中进行。在其他实施方案中，扩增包括在第一扩增反应中采用内侧引物和在第二扩增反应中采用外侧引物，其中第二扩增反应不同于(separate from)第一扩增反应。在这一后者实施方案的变化形式中，第二扩增反应包括两个分别的扩增反应，其中一个扩增反应采用第一外侧引物组且另一个扩增反应采用第二外侧引物组。任选地可汇集在两个分别的第二扩增反应中产生的靶扩增子。In some bidirectional embodiments, amplification is carried out in a single amplified reaction. In other embodiments, amplification comprises adopting inner primers in the first amplified reaction and adopting outer primers in the second amplified reaction, wherein the second amplified reaction is different from (separate from) the first amplified reaction. In the variation of this latter embodiment, the second amplified reaction comprises two amplified reactions respectively, wherein one of the amplified reactions adopts the first outer primer group and another amplified reaction adopts the second outer primer group. Optionally, the target amplicon produced in the two second amplified reactions respectively can be brought together.

在任何上述双向实施方案中，该方法可包括扩增多个靶核酸。多个靶核酸可以是，例如，基因组DNA、cDNA、片段化的DNA、从RNA逆转录的DNA、DNA文库、或从细胞、体液或组织样品提取或扩增的核酸。在具体实施方案中，多个靶核酸是从福尔马林固定的、石蜡包埋的组织样品扩增的。In any of the above-mentioned two-way embodiments, the method may include amplifying multiple target nucleic acids. The multiple target nucleic acids can be, for example, genomic DNA, cDNA, fragmented DNA, DNA reverse transcribed from RNA, a DNA library, or nucleic acids extracted or amplified from a cell, body fluid, or tissue sample. In a specific embodiment, the multiple target nucleic acids are amplified from a formalin-fixed, paraffin-embedded tissue sample.

任何上述双向方法可另外包括测序靶扩增子。例如，当如上述产生的靶扩增子包含另外的核苷酸序列时，该方法可包括利用结合第一和第二另外的核苷酸序列的引物另外扩增以产生用于DNA测序的模板。在具体实施方案中，结合第一和第二另外的核苷酸序列的引物之一或二者被固定在基质(substrate)上。在具体实施方案中，扩增以产生DNA测序模板可通过等温核酸扩增进行。在某些实施方案中，该方法包括利用模板和结合第一和第二引物结合位点并引发靶核苷酸序列的测序的引物进行DNA测序；这些引物优选地以大致上相等的量存在。在一些实施方案中，该方法包括利用模板和结合第一和第二引物结合位点并引发条形码核苷酸序列的测序的引物进行DNA测序；这些引物优选地以大致上相等的量存在。在具体实施方案中，该方法包括利用模板和结合第一和第二引物结合位点并引发条形码核苷酸序列的测序的引物进行DNA测序；其中引物是引发靶核苷酸序列的测序的引物的反向互补物。在示意性实施方案中，用以引发靶核苷酸序列和条形码核苷酸序列的测序的引物包含CS1、CS2、CS1rc和CS2rc(表2，实施例9)。Any of the above-described bidirectional methods may further include sequencing the target amplicon. For example, when the target amplicon generated as described above comprises an additional nucleotide sequence, the method may include further amplification using primers that bind to the first and second additional nucleotide sequences to produce a template for DNA sequencing. In specific embodiments, one or both of the primers that bind to the first and second additional nucleotide sequences are immobilized on a substrate. In specific embodiments, amplification to produce a DNA sequencing template may be performed by isothermal nucleic acid amplification. In certain embodiments, the method includes performing DNA sequencing using the template and primers that bind to the first and second primer binding sites and trigger sequencing of the target nucleotide sequence; these primers are preferably present in substantially equal amounts. In some embodiments, the method includes performing DNA sequencing using the template and primers that bind to the first and second primer binding sites and trigger sequencing of a barcode nucleotide sequence; these primers are preferably present in substantially equal amounts. In specific embodiments, the method includes performing DNA sequencing using the template and primers that bind to the first and second primer binding sites and trigger sequencing of a barcode nucleotide sequence; wherein the primers are the reverse complements of the primers that trigger sequencing of the target nucleotide sequence. In an illustrative embodiment, the primers used to prime the sequencing of the target nucleotide sequence and the barcode nucleotide sequence include CS1, CS2, CS1rc, and CS2rc (Table 2, Example 9).

在任何上述双向实施方案中，可选择条形码核苷酸序列以避免大致上退火(substantial annealing)到靶核酸。在某些实施方案中，条形码核苷酸序列辨识特定样品。In any of the above bidirectional embodiments, the barcode nucleotide sequence can be selected to avoid substantial annealing to the target nucleic acid.In certain embodiments, the barcode nucleotide sequence identifies a specific sample.

在一些实施方案中，当按照上述方法进行双向DNA测序时，从DNA测序确定的序列的至少50％以大于序列的平均拷贝数的50％和小于序列的平均拷贝数的2倍存在。在某些实施方案中，从DNA测序确定的序列的至少70％以大于序列的平均拷贝数的50％和小于序列的平均拷贝数的2倍存在。在具体实施方案中，从DNA测序确定的序列的至少90％以大于序列的平均拷贝数的50％和小于序列的平均拷贝数的2倍存在。In some embodiments, when bidirectional DNA sequencing is performed according to the above method, at least 50% of the sequences determined from the DNA sequencing are present at more than 50% of the average copy number of the sequence and less than 2 times the average copy number of the sequence. In certain embodiments, at least 70% of the sequences determined from the DNA sequencing are present at more than 50% of the average copy number of the sequence and less than 2 times the average copy number of the sequence. In specific embodiments, at least 90% of the sequences determined from the DNA sequencing are present at more than 50% of the average copy number of the sequence and less than 2 times the average copy number of the sequence.

在任何上述双向实施方案中，靶扩增子的平均长度小于200个碱基。在多个实施方案中，第一扩增(即，产生靶扩增子的扩增)在约1皮升至约50纳升或约5皮升至约25纳升的范围的体积中进行。在具体实施方案中，第一扩增(即，产生靶扩增子的扩增)反应在扩增之前在微流体装置的分别的区室(compartment)中形成或被分配到微流体装置的分别的区室中。微流体装置可以是，例如，至少部分地从弹性体材料制造的那些。在某些实施方案中，第一扩增(即，产生靶扩增子的扩增)反应在液滴中进行。In any of the above-described bidirectional embodiments, the average length of the target amplicon is less than 200 bases. In a number of embodiments, the first amplification (i.e., the amplification that produces the target amplicon) is performed in a volume ranging from about 1 picoliter to about 50 nanoliters or from about 5 picoliters to about 25 nanoliters. In specific embodiments, the first amplification (i.e., the amplification that produces the target amplicon) reaction is formed in or distributed to separate compartments of the microfluidic device prior to amplification. The microfluidic device can be, for example, those manufactured at least in part from an elastomeric material. In certain embodiments, the first amplification (i.e., the amplification that produces the target amplicon) reaction is performed in a droplet.

本发明的另一方面包括可用于进行以上讨论的双向实施方案的试剂盒。在某些实施方案中，试剂盒包含：Another aspect of the present invention includes a kit that can be used to perform the bidirectional embodiment discussed above. In certain embodiments, the kit comprises:

第一外侧引物，其中该组包含：First outer primers, wherein the set comprises:

第一外侧、正向引物，包含对第一引物结合位点特异性的部分；和a first outer, forward primer comprising a portion specific for the first primer binding site; and

第一外侧、反向引物，包含条形码核苷酸序列和对第二引物结合位点特异性的部分，其中第一和第二引物结合位点是不同的；a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for a second primer binding site, wherein the first and second primer binding sites are different;

第二外侧引物组，其中该组包含：a second outer primer set, wherein the set comprises:

第二外侧、正向引物，包含条形码核苷酸序列和对第一引物结合位点特异性的部分；和a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and

第二外侧、反向引物，包含对第二引物结合位点特异性的部分。在具体实施方案中，第一和第二引物结合位点是DNA测序引物的结合位点。在具体实施方案中，外侧引物各自另外包含另外的核苷酸序列，其中：The second outer, reverse primer comprises a portion specific for the second primer binding site. In a specific embodiment, the first and second primer binding sites are binding sites for DNA sequencing primers. In a specific embodiment, each of the outer primers further comprises an additional nucleotide sequence, wherein:

第一外侧、正向引物包含第一另外的核苷酸序列，且第一外侧、反向引物包含第二另外的核苷酸序列；和The first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and

第二外侧、正向引物包含第二另外的核苷酸序列，且第二外侧、反向引物包含第一另外的核苷酸序列，且第一和第二另外的核苷酸序列是不同的。在示例性实施方案中，第一外侧引物组包含PE1-CS1和PE2-BC-CS2，且第二外侧引物组包含PE1-CS2和PE2-BC-CS1(表1，实施例9)。在某些实施方案中，试剂盒另外包含内侧引物组，其中该组包含：The second outer, forward primer comprises a second additional nucleotide sequence, and the second outer, reverse primer comprises a first additional nucleotide sequence, and the first and second additional nucleotide sequences are different. In an exemplary embodiment, the first outer primer set comprises PE1-CS1 and PE2-BC-CS2, and the second outer primer set comprises PE1-CS2 and PE2-BC-CS1 (Table 1, Example 9). In certain embodiments, the kit further comprises an inner primer set, wherein the set comprises:

内侧、正向引物，包含靶特异性部分和第一引物结合位点；和an inner, forward primer comprising a target-specific portion and a first primer binding site; and

内侧、反向引物，包含靶特异性部分和第二引物结合位点。在一些实施方案中，试剂盒包含各自对不同的靶核酸特异性的多个内侧引物组。An inner, reverse primer comprising a target-specific portion and a second primer binding site. In some embodiments, the kit comprises a plurality of inner primer sets, each specific for a different target nucleic acid.

可用于进行双向实施方案的任何上述试剂盒可另外包含结合第一和第二引物结合位点并引发靶核苷酸序列的测序的DNA测序引物和/或另外包含结合第一和第二引物结合位点并引发条形码核苷酸序列的测序的DNA测序引物。在具体实施方案中，结合第一和第二引物结合位点并引发条形码核苷酸序列的测序的引物是引发靶核苷酸序列的测序的引物的反向互补物。例如，用以引发靶核苷酸序列和条形码核苷酸序列的测序的引物包含CS1、CS2、CS1rc和CS2rc(表2，实施例9)。Any of the above kits that can be used to perform the bidirectional embodiment can additionally comprise DNA sequencing primers that bind to the first and second primer binding sites and initiate sequencing of the target nucleotide sequence and/or further comprise DNA sequencing primers that bind to the first and second primer binding sites and initiate sequencing of the barcode nucleotide sequence. In specific embodiments, the primers that bind to the first and second primer binding sites and initiate sequencing of the barcode nucleotide sequence are the reverse complements of the primers that initiate sequencing of the target nucleotide sequence. For example, the primers used to initiate sequencing of the target nucleotide sequence and the barcode nucleotide sequence include CS1, CS2, CS1rc, and CS2rc (Table 2, Example 9).

在一些实施方案中，本发明还提供检测、和/或定量核酸样品中至少两个不同的靶核酸的相对量的方法。该方法包括从样品中的第一和第二靶核酸产生第一和第二加标签的靶核苷酸序列，In some embodiments, the present invention also provides a method for detecting and/or quantifying the relative amounts of at least two different target nucleic acids in a nucleic acid sample. The method comprises generating first and second tagged target nucleotide sequences from the first and second target nucleic acids in the sample,

第一加标签的靶核苷酸序列包含第一核苷酸标签；和the first tagged target nucleotide sequence comprises a first nucleotide tag; and

第二加标签的靶核苷酸序列包含第二核苷酸标签，其中第一和第二核苷酸标签是不同的。对加标签的靶核苷酸序列进行利用退火到第一核苷酸标签的第一引物的第一引物延伸反应，和利用退火到第二核苷酸标签的第二引物的第二引物延伸反应。该方法还包括检测和/或定量指示第一引物的延伸的信号、和指示第二引物的延伸的信号，其中给定引物的信号指示相应的靶核酸的存在、和/或相对量。The second tagged target nucleotide sequence comprises a second nucleotide tag, wherein the first and second nucleotide tags are different. The tagged target nucleotide sequence is subjected to a first primer extension reaction using a first primer that anneals to the first nucleotide tag, and a second primer extension reaction using a second primer that anneals to the second nucleotide tag. The method further includes detecting and/or quantifying a signal indicative of extension of the first primer and a signal indicative of extension of the second primer, wherein the signal for a given primer indicates the presence and/or relative amount of the corresponding target nucleic acid.

附图简述BRIEF DESCRIPTION OF THE DRAWINGS

图1A-1D：发夹衔接子分子产生衔接子修饰的靶核酸分子例如适合用于高通量DNA测序的文库的图解。(A)发夹衔接子分子，各自包含：衔接子核苷酸序列，其连接于核苷酸接头，其连接于核苷酸序列，其能够退火到衔接子核苷酸序列并连接于简并尾序列；N＝核苷酸；任选的特异性酶切割位点可被包含在核苷酸接头中。(B)靶核酸分子制备可包括片段化和消化5'末端以产生3'粘末端。(C)进行退火、填充缺口、和连接。(D)利用在接头内切割的酶方便地将所得的DNA线性化。Figures 1A-1D: Schematic illustration of hairpin adapter molecules generating adapter-modified target nucleic acid molecules, such as libraries suitable for high-throughput DNA sequencing. (A) Hairpin adapter molecules, each comprising: an adapter nucleotide sequence linked to a nucleotide linker linked to a nucleotide sequence capable of annealing to the adapter nucleotide sequence and ligating to a degenerate tail sequence; N = nucleotide; an optional specific enzyme cleavage site may be included in the nucleotide linker. (B) Target nucleic acid molecule preparation may include fragmentation and digestion of the 5' end to generate 3' sticky ends. (C) Annealing, gap filling, and ligation are performed. (D) The resulting DNA is conveniently linearized using an enzyme that cleaves within the linker.

图2A-2D：双链衔接子分子产生衔接子修饰的靶核酸分子例如适合用于高通量DNA测序的文库的图解。(A)双链衔接子分子，在每条链上各自包含：第一衔接子核苷酸序列，其连接于核苷酸接头，其连接于第二衔接子核苷酸序列；和简并尾序列，其中双链分子各自包含两个简并尾序列作为粘末端；N＝核苷酸；任选的特异性酶切割位点可被包含在核苷酸接头中。(B)靶核酸分子制备可包括片段化和消化5'末端以产生3'粘末端。(C)进行退火、填充缺口、和连接。(D)利用在接头内切割的酶方便地将所得的环状DNA线性化。Figures 2A-2D: Schematic diagram of double-stranded adapter molecules generating adapter-modified target nucleic acid molecules, such as libraries suitable for high-throughput DNA sequencing. (A) Double-stranded adapter molecules, each comprising on each strand: a first adapter nucleotide sequence, which is linked to a nucleotide linker, which is linked to a second adapter nucleotide sequence; and a degenerate tail sequence, wherein each double-stranded molecule comprises two degenerate tail sequences as sticky ends; N = nucleotide; optional specific enzyme cleavage sites can be included in the nucleotide linker. (B) Target nucleic acid molecule preparation can include fragmentation and digestion of the 5' end to generate 3' sticky ends. (C) Annealing, gap filling, and ligation are performed. (D) The resulting circular DNA is conveniently linearized using an enzyme that cuts within the linker.

图3：四引物、组合条形码化方法可用于将两个条形码的组合放置在每个扩增子的任一末端上。内侧引物包含靶特异性部分(正向引物中的"TS-F"和反向引物中的"TS-R")、条形码核苷酸序列("bc2")、和不同的核苷酸标签。外侧引物包含标签特异性部分("CS1"和"CS2")、不同的条形码核苷酸序列("bc1")、用于测序引物的引物结合位点("A"和"B")。Figure 3: A four-primer, combinatorial barcoding approach can be used to place a combination of two barcodes on either end of each amplicon. The inner primers contain target-specific portions ("TS-F" in the forward primer and "TS-R" in the reverse primer), a barcode nucleotide sequence ("bc2"), and different nucleotide tags. The outer primers contain tag-specific portions ("CS1" and "CS2"), different barcode nucleotide sequences ("bc1"), and primer binding sites for sequencing primers ("A" and "B").

图4：六引物、组合条形码化方法可用于将两个条形码的组合放置在每个扩增子的任一末端上。内侧引物包含靶特异性部分(正向引物中的"TS-F"和反向引物中的"TS-R")和不同的核苷酸标签。填充引物包含标签特异性部分("CS1"和"CS2")、条形码核苷酸序列("bc2")、和两个另外的不同的核苷酸标签。外侧引物包含对两个另外的核苷酸标签特异性的部分("CS3"和"CS4")、不同的条形码核苷酸序列("bc1")、和用于测序引物的引物结合位点("A"和"B")。Figure 4: A six-primer, combinatorial barcoding approach can be used to place a combination of two barcodes on either end of each amplicon. The inner primers contain target-specific portions ("TS-F" in the forward primer and "TS-R" in the reverse primer) and different nucleotide tags. The filler primers contain tag-specific portions ("CS1" and "CS2"), a barcode nucleotide sequence ("bc2"), and two additional different nucleotide tags. The outer primers contain portions specific for two additional nucleotide tags ("CS3" and "CS4"), a different barcode nucleotide sequence ("bc1"), and primer binding sites for sequencing primers ("A" and "B").

图5A-5B：基于组合连接的加标签方法(combinatorial ligation-based taggingmethod)利用加标签的靶核苷酸序列(A)以产生组合加标签的靶核苷酸序列。PE1、PE1＝Illumina测序流动小室(flowcell)结合序列；Seq1、Seq2＝测序引发位点；BC1、BC2＝条形码序列。参见实施例2。FIG5A-5B : Combinatorial ligation-based tagging method utilizes tagged target nucleotide sequences (A) to generate combinatorially tagged target nucleotide sequences. PE1, PE1 = Illumina sequencing flow cell binding sequence; Seq1, Seq2 = sequencing priming sites; BC1, BC2 = barcode sequences. See Example 2.

图6：用于测序(例如，Illumina测序)的基于组合插入诱变的加标签(combinatorial insertional mutagenesis-based tagging)。将条形码插入到转座子标签序列中。TagA和TagB需要足够长来引发测序。BC2应包含4碱基的条形码加在5'末端的3个简并引物(例如NNNAGTC)。转座子末端序列＝5'-AGATGTGTATAAGAGACAG-3'(SEQ ID NO：1)。PE1、PE1＝Illumina测序流动小室结合序列；BC1、BC2＝条形码序列。Figure 6: Combinatorial insertional mutagenesis-based tagging for sequencing (e.g., Illumina sequencing). The barcode is inserted into the transposon tag sequence. TagA and TagB need to be long enough to prime sequencing. BC2 should contain three degenerate primers with a 4-base barcode added to the 5' end (e.g., NNNAGTC). Transposon end sequence = 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 1). PE1, PE1 = Illumina sequencing flow cell binding sequence; BC1, BC2 = barcode sequence.

图7A-C：条形码化和汇集反应混合物用于后续分析：条形码化的靶核苷酸序列的产生。(A)在示例性实施方案中，将细胞以有限稀释上样到ACCESS ARRAY^TM IFC("Integrated Fluidic Circuit"，在本文还称为"芯片")中。如所示地上样引物组，芯片中的每个室(chamber)接收完整组的96个正向引物(F1-96)和96个反向引物(R1-96)用于扩增96个靶。反向引物用能退火到条形码引物的标签加标签。芯片的行中的每个室接收不同的条形码引物。(B)如实施例5中所述的，利用3引物方法在芯片中进行逆转录和预扩增以产生条形码化的靶核苷酸序列。任何指定的室将扩增所有基因，且所有扩增子将被单个条形码加标签。反应产物由池(与不同的引物组成90度，即由样品)输出。(C)对于检测，可如所示地上样DYNAMIC ARRAY^TM IFC，正向引物(例如，F1)用于扩增特定靶核酸且条形码引物(例如，BC1)用于扩增特定池(例如池1)的特定室中的这一序列。Figure 7A-C: Barcoding and pooling of reaction mixtures for subsequent analysis: Generation of barcoded target nucleotide sequences. (A) In an exemplary embodiment, cells are loaded into an ACCESS ARRAY^™ IFC ("Integrated Fluidic Circuit", also referred to herein as a "chip") at limiting dilution. Primer sets are loaded as shown, with each chamber in the chip receiving a complete set of 96 forward primers (F1-96) and 96 reverse primers (R1-96) for amplifying 96 targets. The reverse primers are tagged with tags that can anneal to the barcode primers. Each chamber in a row of the chip receives a different barcode primer. (B) As described in Example 5, reverse transcription and pre-amplification are performed in the chip using a 3-primer approach to generate barcoded target nucleotide sequences. Any given chamber will amplify all genes, and all amplicons will be tagged with a single barcode. Reaction products are output by a pool (90 degrees from the different primers, i.e., by sample). (C) For detection, a DYNAMIC ARRAY^™ IFC can be loaded as shown, a forward primer (eg, F1) used to amplify a specific target nucleic acid and a barcode primer (eg, BC1) used to amplify this sequence in a specific chamber of a specific pool (eg, Pool 1).

图8A-C：条形码化和汇集反应混合物用于后续分析：扩增/检测条形码化的靶核苷酸序列的示例性策略。(A)示例性实施方案利用LCR检测具有以下结构的条形码化的靶核苷酸序列：5'-正向引物序列-靶核苷酸序列-反向引物序列-条形码核苷酸序列-3'。在这种情形中，一个引物可退火到反向引物序列，另一个引物可退火到邻近的条形码核苷酸序列，随后是连接、和重复的退火和连接循环。(B)检测可利用悬垂片(flap)核酸内切酶-连接酶链式反应实时进行。这一反应采用标记的探针和未标记的探针，其中探针对反应产物的同时杂交导致在标记的探针的5'末端形成悬垂片，且悬垂片的裂解产生信号。如所示的，悬垂片的裂解可分离荧光团与猝灭剂以产生信号。(C)可用于，例如，检测通过LCR从具有以下结构的条形码化的靶核苷酸序列产生的扩增子的替代性实时检测方法：5'-正向引物序列-靶核苷酸序列-反向引物序列-条形码核苷酸序列-3'。这一方法依赖于使用双链DNA结合染料(dye)来检测反应产物和LCR所用的引物之间的解链温度差异。解链温度分析包括在反应产物是大致上双链且在双链DNA结合染料存在下能够产生信号、但引物是大致上单链且不能产生信号的温度("高温")下的检测。例如，对于检测具有以下结构的条形码化的靶核苷酸序列：5'-正向引物序列-靶核苷酸序列-反向引物序列-条形码核苷酸序列-3'，一个引物可退火到反向引物序列，另一个引物可退火到邻近的条形码核苷酸序列，随后是连接、和重复的退火和连接循环。参见图8C。Figure 8A-C: Barcoding and pooling of reaction mixtures for subsequent analysis: Exemplary strategies for amplifying/detecting barcoded target nucleotide sequences. (A) An exemplary embodiment utilizes LCR to detect a barcoded target nucleotide sequence having the following structure: 5'-forward primer sequence-target nucleotide sequence-reverse primer sequence-barcode nucleotide sequence-3'. In this scenario, one primer can anneal to the reverse primer sequence and the other primer can anneal to the adjacent barcode nucleotide sequence, followed by ligation, and repeated cycles of annealing and ligation. (B) Detection can be performed in real time using a flap endonuclease-ligase chain reaction. This reaction employs a labeled probe and an unlabeled probe, wherein simultaneous hybridization of the probes to the reaction products results in the formation of a flap at the 5' end of the labeled probe, and cleavage of the flap generates a signal. As shown, cleavage of the flap can separate the fluorophore from the quencher to generate a signal. (C) An alternative real-time detection method that can be used, for example, to detect amplicons generated by LCR from a barcoded target nucleotide sequence having the following structure: 5'-forward primer sequence-target nucleotide sequence-reverse primer sequence-barcode nucleotide sequence-3'. This method relies on the use of a double-stranded DNA binding dye to detect the difference in melting temperature between the reaction product and the primers used for LCR. Melting temperature analysis includes detection at a temperature ("high temperature") at which the reaction product is substantially double-stranded and can produce a signal in the presence of a double-stranded DNA binding dye, but the primer is substantially single-stranded and cannot produce a signal. For example, for detecting a barcoded target nucleotide sequence having the following structure: 5'-forward primer sequence-target nucleotide sequence-reverse primer sequence-barcode nucleotide sequence-3', one primer can anneal to the reverse primer sequence and the other primer can anneal to the adjacent barcode nucleotide sequence, followed by ligation and repeated annealing and ligation cycles. See Figure 8C.

图9：用于适于细胞操作的微流体装置的单元小室结构("MA006")的示意图，显示芯片上方法(on-chip processes)。Figure 9: Schematic diagram of the unit cell structure ("MA006") for a microfluidic device suitable for cell manipulation, showing on-chip processes.

图10：使用细胞悬液的有限稀释来获得每单独反应体积(微流体装置的"室"或"芯片")单个细胞。显示了用于不同细胞密度的理论分布(泊松分布)。Figure 10: Limiting dilution of a cell suspension is used to obtain a single cell per individual reaction volume (a "chamber" or "chip" of a microfluidic device). Theoretical distributions (Poisson distribution) for different cell densities are shown.

图11 A-B：使用亮视野(A)成像，芯片中细胞计数的结果，与理论分布(B)比较。基于亮视野成像，芯片中的细胞密度接近但低于泊松分布，这一趋势在更高细胞密度时加剧。Figure 11 A-B: Cell count results in the chip using bright-field imaging (A), compared with the theoretical distribution (B). Based on bright-field imaging, the cell density in the chip is close to but lower than the Poisson distribution, and this trend is exacerbated at higher cell densities.

图12A-B：荧光细胞"鬼影(ghost)"图像(A)允许比PCR前(pre-PCR)亮视野成像检测更多细胞，从而细胞密度更接近地近似泊松分布(B)。12A-B: Fluorescent cell "ghost" images (A) allow detection of more cells than pre-PCR bright-field imaging, resulting in cell density more closely approximating a Poisson distribution (B).

图13：可以使用的检测芯片中细胞的特异性方法包括，例如，使用细胞膜透性核酸染色剂(stain)和/或用抗体的细胞特异性表面标志物检测。这些更特异性的方法的结果对1E6/ml的细胞密度显示。Figure 13: Specific methods that can be used to detect cells in the chip include, for example, the use of cell membrane permeable nucleic acid stains and/or cell-specific surface marker detection using antibodies. Results from these more specific methods are shown for a cell density of 1E6/ml.

图14A-B：(A)RT-PCR前(pre-RT-PCR)核酸染色剂(Syto10 DNA染色剂)检测芯片中细胞与RT-PCR后(post RT-PCR)鬼影图像(细胞鬼影)的比较。(B)Syto10不抑制GAPDH的RT-PCR。Figure 14A-B: (A) Comparison of pre-RT-PCR (pre-RT-PCR) nucleic acid stain (Syto10 DNA stain) detection of cells in the chip and post-RT-PCR (cell ghost) images. (B) Syto10 does not inhibit GAPDH RT-PCR.

图15：在0.5％Tween 20或0.5％NP40(后者是细胞溶解剂)存在下进行的GAPDH的RT-PCR。二者都不显著抑制GAPDH的RT-PCR。Figure 15: RT-PCR of GAPDH performed in the presence of 0.5% Tween 20 or 0.5% NP40 (the latter is a cell lysis agent). Neither of them significantly inhibited RT-PCR of GAPDH.

图16：在MA006芯片中进行的11个基因的标准曲线扩增。这些结果证明，CellsDirect^TM一步qRT-PCR试剂盒可与0.5％NP40(用于细胞溶解和阻止芯片中的耗竭效应)一起使用来在MA006芯片中将细胞中基因特异性的RNA转化为扩增子。Figure 16: Standard curve amplification of 11 genes in the MA006 chip. These results demonstrate that the CellsDirect^™ One-Step qRT-PCR Kit can be used with 0.5% NP40 (for cell lysis and prevention of on-chip depletion effects) to convert gene-specific RNA from cells into amplicons in the MA006 chip.

图17：四引物、组合条形码化方法用以将两个条形码的组合放到每个扩增子的任一末端上。内侧引物包含靶特异性部分(正向引物中的"TS-F"和反向引物中的"TS-R")、条形码核苷酸序列("bc2")、和不同的核苷酸标签。外侧引物包含标签特异性部分("CS1"和"CS2")、不同的条形码核苷酸序列("bc1")、用于测序引物的引物结合位点("A"和"B")。Figure 17: A four-primer, combinatorial barcoding approach is used to place a combination of two barcodes on either end of each amplicon. The inner primers contain target-specific portions ("TS-F" in the forward primer and "TS-R" in the reverse primer), a barcode nucleotide sequence ("bc2"), and different nucleotide tags. The outer primers contain tag-specific portions ("CS1" and "CS2"), different barcode nucleotide sequences ("bc1"), and primer binding sites for sequencing primers ("A" and "B").

图18A-B：4引物条形码化如何可在芯片诸如MA006上进行的图解。(A)在芯片上用内侧引物进行扩增，其中每行的室具有拥有相同的条形码的相同的内侧引物对。(B)来自每列的室的反应产物可作为池收获，并对每个池利用不同的外侧引物对进行扩增。这一扩增产生在扩增子的任一末端具有独特地辨识在其中进行初始扩增的室(以行和列)的条形码组合的扩增子。Figure 18A-B: Schematic illustration of how 4-primer barcoding can be performed on a chip such as the MA006. (A) Amplification is performed on the chip using inner primers, where each row of chambers has the same inner primer pair with the same barcode. (B) Reaction products from each column of chambers can be harvested as a pool and amplified using a different outer primer pair for each pool. This amplification produces amplicons with a barcode combination at either end that uniquely identifies the chamber (row and column) in which the initial amplification was performed.

图19：比较测序来自单细胞的基因特异性扩增子获得的结果(实施例5)，表示为对每个基因特异性扩增子的读取的数目，与总RNA的比较。如从该图明显的，这些RNA的代表当在单独细胞中测量时是不同的，与在总RNA中观察到的相比。Figure 19: Comparison of the results obtained from sequencing gene-specific amplicons from single cells (Example 5), expressed as the number of reads for each gene-specific amplicon, compared to total RNA. As is apparent from this figure, the representation of these RNAs is different when measured in individual cells than when observed in total RNA.

图20 A-B：带有捕获特征(capture feature)和引流沟(drain)的捕获位点。(A)无阻碍物(baffle)以集中流(flow)的位点。(B)带有阻碍物的位点。Figure 20 A-B: Capture sites with capture features and drains. (A) Site without baffles to focus flow. (B) Site with baffles.

图21：另外的捕获位点设计。Figure 21: Additional capture site designs.

图22A-C：可设计捕获结构(capture architecture)以使细胞接触表面标志物的可能性最大化。例如，一个或多个通道壁(channel wall)上的阻碍物可用于引导珠朝向捕获特征。(A)示例性的捕获特征/阻碍物组合。(B)捕获特征的表现可通过调整一个或多个变量来调整，所述变量包括阻碍物的角度、阻碍物与捕获位点的距离、阻碍物的长度、捕获特征的尺寸和形状、捕获特征中引流沟的尺寸(如果存在)。通道壁上的阻碍物用于引导珠朝向捕获特征。(C)捕获特征与通道壁上的阻碍物配对；单独的捕获特征/阻碍物组合可位于交替的壁上以将流集中朝向邻近的捕获特征/阻碍物组合。Figure 22A-C: Capture architecture can be designed to maximize the likelihood that cells will contact surface markers. For example, obstructions on one or more channel walls can be used to guide beads toward capture features. (A) Exemplary capture feature/obstruction combinations. (B) The performance of a capture feature can be tuned by adjusting one or more variables, including the angle of the obstruction, the distance of the obstruction from the capture site, the length of the obstruction, the size and shape of the capture feature, and the size of the drainage groove in the capture feature (if present). Obstructions on the channel walls are used to guide beads toward the capture feature. (C) Capture features are paired with obstructions on the channel walls; separate capture feature/obstruction combinations can be located on alternating walls to focus flow toward adjacent capture feature/obstruction combinations.

图23 A-B：利用捕获特征来捕捉单个、包被亲和性试剂的珠的策略，该珠随后展示亲和性试剂(例如，抗体)以捕获单颗粒(例如，细胞)。(A-1)流在包含捕获特征的通道中开始。(A-2)抗体结合的珠流向捕获特征，直到珠安顿在捕获特征中。(A-3)然后洗涤通道以去除未捕获的珠。(B-1)带有抗体结合的细胞表面标志物的细胞流入包含捕获的珠的通道。(B-2)带有标志物的细胞与被捕获的珠展示的抗体相互作用和结合。展示区域的尺寸为使得结合的细胞将经由空间阻塞抑制其他细胞与捕获的珠相互作用，从而仅一个细胞结合每个捕获的珠。(B-3)然后洗涤通道以去除未结合的细胞，在每个捕获位点留下一个固定的细胞。Figure 23 A-B: Strategy for utilizing capture features to capture single, affinity-reagent-coated beads, which then display an affinity reagent (e.g., an antibody) to capture single particles (e.g., cells). (A-1) Flow begins in a channel containing a capture feature. (A-2) Antibody-bound beads flow toward the capture feature until the beads settle into the capture feature. (A-3) The channel is then washed to remove uncaptured beads. (B-1) Cells bearing antibody-bound cell surface markers flow into a channel containing captured beads. (B-2) The labeled cells interact and bind to the antibodies displayed by the captured beads. The display area is sized such that bound cells will inhibit other cells from interacting with the captured beads via steric blockage, resulting in only one cell binding to each captured bead. (B-3) The channel is then washed to remove unbound cells, leaving one immobilized cell at each capture site.

图24A-G：(A)设计为在离散的位置(龛(niches))捕获单细胞的微流体装置的简图。单细胞捕获允许在单细胞水平分析生物事件。(B)流设计为在龛上方比经过溢流通道的强。龛包含小的缺口(～3μm高)。当细胞进入龛时，它封闭龛并阻止任何更多的流进入龛。流穿过到下一个未被占据的龛，直到它也被细胞封闭。在细胞穿过溢流通道并离开废弃(outto waste)之前，每个龛应捕获一个细胞。(C)显示带有(D)-(F)中提供的另外的细节的(A)的简图。(D)缓冲液入口与细胞入口汇集(converge)，从而迫使细胞向最接近一系列横向细胞捕获通道(transverse cell capture channel)的给料通道的一侧。(E)横向细胞捕获通道的阻力低于细胞溢流通道的，以引导细胞流优先进入龛而不是进入细胞溢流通道。(F)每个龛足够大以捕获仅一个细胞。龛中的细胞升高了该特定回路的阻力，流被导向无细胞的回路。(G)(A)的实际装置，捕获的人类脐静脉内皮细胞(HUVEC)位于龛中。Figure 24A-G: (A) Schematic diagram of a microfluidic device designed to capture single cells at discrete locations (niches). Single-cell capture allows analysis of biological events at the single-cell level. (B) Flow is designed to be stronger over the niches than through the overflow channels. Niches contain small gaps (~3 μm high). When a cell enters a niche, it seals it and prevents further flow from entering. Flow passes to the next unoccupied niche until it is also sealed by a cell. Each niche should capture a single cell before the cell passes through the overflow channels and out to waste. (C) Schematic diagram showing (A) with additional details provided in (D)-(F). (D) The buffer inlet converges with the cell inlet, forcing the cell toward the side of the feed channel closest to a series of transverse cell capture channels. (E) The transverse cell capture channels have lower resistance than the cell overflow channels, directing cell flow preferentially into the niches rather than into the cell overflow channels. (F) Each niche is large enough to capture only a single cell. Cells in the niche increase the resistance of that particular circuit, and flow is directed toward the cell-free circuit. (G) Actual device from (A), with captured human umbilical vein endothelial cells (HUVECs) located in the niche.

图25 A-B：实施例9中采用的扩增子加标签策略。(A)标准4引物扩增子加标签相对于双向测序扩增子加标签。标准4引物扩增子加标签方法在共有序列标签1(CS1)和共有序列标签2(CS2)中掺入了末端配对的Illumina测序引物退火位点。每个PCR产物的5'末端和3'末端的测序均要求末端配对的测序运行。(B)靶特异性引物附带有共有序列标签CS1和CS2。样品特异性引物对包含共有序列标签CS1或CS2，以两种排列附带有Genome Analyzer所用的衔接子序列(PE1和PE2)。从相同的靶区域产生两个PCR产物类型：在相同的测序读取中，产物A允许测序靶区域的5'末端而产物B允许测序靶区域的3'末端。Figure 25 A-B: Amplicon tagging strategy employed in Example 9. (A) Standard 4-primer amplicon tagging versus bidirectional sequencing amplicon tagging. The standard 4-primer amplicon tagging method incorporates paired-end Illumina sequencing primer annealing sites in consensus sequence tag 1 (CS1) and consensus sequence tag 2 (CS2). Sequencing of the 5' and 3' ends of each PCR product requires a paired-end sequencing run. (B) Target-specific primers are accompanied by consensus sequence tags CS1 and CS2. Sample-specific primer pairs contain either consensus sequence tags CS1 or CS2, accompanied by adapter sequences (PE1 and PE2) for use with the Genome Analyzer in two arrangements. Two PCR product types are generated from the same target region: Product A allows sequencing of the 5' end of the target region and Product B allows sequencing of the 3' end of the target region in the same sequencing read.

图26A-B：实施例9中使用的分离的-引物PCR(segregated-primer PCR)策略的概述。用靶特异性引物对的第一PCR在ACCESS ARRAY^TM IFC中进行。将收获的PCR产物池分为用样品特异性条形码引物的两个随后PCR反应。(A)产生允许测序靶区域5'末端的产物的反应利用PE1_CS1和PE2_BC_CS2引物组合。(B)产生允许测序靶区域3'末端的产物的反应利用PE1_CS2和PE2_BC_CS1引物组合。Figure 26A-B: Overview of the segregated-primer PCR strategy used in Example 9. The first PCR using target-specific primer pairs was performed in the ACCESS ARRAY^™ IFC. The harvested PCR product pool was split into two subsequent PCR reactions using sample-specific barcoded primers. (A) The reaction that generated a product that allowed sequencing of the 5' end of the target region utilized the PE1_CS1 and PE2_BC_CS2 primer combinations. (B) The reaction that generated a product that allowed sequencing of the 3' end of the target region utilized the PE1_CS2 and PE2_BC_CS1 primer combinations.

图27：实施例9中使用的测序工作流程的概述。两个PCR产物类型都存在于流动小室上。CS1和CS2的等摩尔混合物允许测序靶区域的5'末端和3'末端。剥除(stripping)和用CS1rc和CS2rc的等摩尔混合物再杂交簇(cluster)后，测序条形码。测序引物CS1和CS2在试剂FL1中提供。索引引物CS1rc和CS2rc在试剂FL2中提供。Figure 27: Overview of the sequencing workflow used in Example 9. Both PCR product types are present on the flow cell. An equimolar mixture of CS1 and CS2 allows sequencing of the 5' and 3' ends of the target region. After stripping and rehybridizing the cluster with an equimolar mixture of CS1rc and CS2rc, the barcodes are sequenced. Sequencing primers CS1 and CS2 are provided in reagent FL1. Index primers CS1rc and CS2rc are provided in reagent FL2.

图28：用来自实施例10中的板1和板2的条形码从条形码化反应运行获得的Bioanalyzer产物。Figure 28: Bioanalyzer products obtained from a barcoding reaction run using barcodes from plates 1 and 2 in Example 10.

图29：实施例10中使用的替代的测序引物。使用ACCESS ARRAY^TMIFC上使用的所有靶特异性PCR引物的等摩尔混合物作为测序引物池避免了经过不提供信息的靶特异性引物区测序。Figure 29: Alternative sequencing primers used in Example 10. Using an equimolar mixture of all target-specific PCR primers used on the ACCESS ARRAY^™ IFC as a sequencing primer pool avoids sequencing through uninformative target-specific primer regions.

图30：基因EGFR对实施例10中一个样品的逐个碱基覆盖。来自每个链的读取以不同的阴影显示。Figure 30: Base-by-base coverage of the gene EGFR for a sample from Example 10. Reads from each strand are shown in different shading.

图31 A-B：(A)在454测序乳液PCR反应之前，在一个反应中进行对靶DNA的等位基因特异性PCR。正向引物具有454个衔接子和等位基因特异性标签。不同的标签以不同的阴影显示。这一反应产生为454珠乳液PCR准备的扩增子。(B)在乳液PCR和上样到测序仪后，每个孔中单独珠上的扩增子是野生型或突变体。第一454循环流动结合野生型标签(粉色箭头)的引物，且流动所有dNTP。随着这一引物延伸，掺入了多个核苷酸，得到非常牢固的信号，但仅在带有野生型分子的孔中。第二循环流动所有dNTP和突变体标签的引物，仅在带有突变体分子的孔中产生信号。Figure 31 A-B: (A) Allele-specific PCR of the target DNA is performed in one reaction prior to the 454 sequencing emulsion PCR reaction. The forward primer has a 454 adapter and an allele-specific tag. Different tags are shown in different shades. This reaction produces amplicons ready for 454 bead emulsion PCR. (B) After emulsion PCR and loading onto the sequencer, the amplicons on the individual beads in each well are either wild type or mutant. The first 454 cycle flows a primer that binds the wild type tag (pink arrow) and flows all dNTPs. As this primer is extended, multiple nucleotides are incorporated, resulting in a very strong signal, but only in wells with wild type molecules. The second cycle flows all dNTPs and a primer with a mutant tag, producing signal only in wells with mutant molecules.

图32：来自Fluidigm和Illumina TruSeq测序引物之间对Illumina产生的文库的相互干扰实验的Agilent Bioanalyzer结果。每个泳道的PCR反应如下：Figure 32: Agilent Bioanalyzer results from an experiment to investigate the mutual interference between Fluidigm and Illumina TruSeq sequencing primers on an Illumina-generated library. The PCR reaction for each lane was as follows:

1.Illumina标准文库+Fluidigm FL1测序引物1.Illumina standard library + Fluidigm FL1 sequencing primer

2.Illumina标准文库+Illumina TruSeq测序引物2.Illumina standard library + Illumina TruSeq sequencing primers

3.Illumina标准文库+Fluidigm FL1和Illumina TruSeq测序引物3. Illumina standard library + Fluidigm FL1 and Illumina TruSeq sequencing primers

4.Illumina标准文库+Illumina标准测序引物(对照)4. Illumina standard library + Illumina standard sequencing primer (control)

5.Illumina多路文库+Fluidigm FL1测序引物5.Illumina multiplex library + Fluidigm FL1 sequencing primer

6.Illumina多路文库+Illumina TruSeq测序引物6. Illumina multiplex library + Illumina TruSeq sequencing primers

7.Illumina多路文库+Fluidigm FL1和Illumina TruSeq测序引物7. Illumina multiplex library + Fluidigm FL1 and Illumina TruSeq sequencing primers

8.Illumina多路文库+Illumina多路测序引物(对照)8. Illumina multiplex library + Illumina multiplex sequencing primers (control)

9.Illumina小RNA文库+Fluidigm FL1测序引物9.Illumina small RNA library + Fluidigm FL1 sequencing primer

10.Illumina小RNA文库+Illumina TruSeq测序引物10.Illumina small RNA library + Illumina TruSeq sequencing primers

11.Illumina小RNA文库+Fluidigm FL1和Illumina TruSeq测序引物11. Illumina small RNA library + Fluidigm FL1 and Illumina TruSeq sequencing primers

12.Illumina小RNA文库+Illumina小RNA测序引物(对照)12. Illumina small RNA library + Illumina small RNA sequencing primers (control)

图33：来自Fluidigm和Illumina TruSeq测序引物之间对ACCESS ARRAY^TM IFC产生的文库的相互干扰实验的Agilent Bioanalyzer结果。每个泳道的PCR反应如下：Figure 33: Agilent Bioanalyzer results from a crosstalk experiment between Fluidigm and Illumina TruSeq sequencing primers on an ACCESS ARRAY^™ IFC generated library. The PCR reaction for each lane was as follows:

1.Fluidigm ACCESS ARRAY^TM IFC文库+Fluidigm FL 1测序引物1. Fluidigm ACCESS ARRAY^™ IFC library + Fluidigm FL 1 sequencing primer

2.Fluidigm ACCESS ARRAY^TM IFC文库+Illumina TruSeq测序引物2. Fluidigm ACCESS ARRAY^™ IFC library + Illumina TruSeq sequencing primers

3.Fluidigm ACCESS ARRAY^TM IFC文库+Fluidigm FL 1和IlluminaTruSeq测序引物3. Fluidigm ACCESS ARRAY^™ IFC library + Fluidigm FL 1 and Illumina TruSeq sequencing primers

详述Details

对于多个应用，掺入核酸序列到例如来源于样品诸如生物样品的靶核酸中是必要或期望的。在某些实施方案中，掺入的序列可帮助进一步分析靶核酸。因此，本文描述了可用于掺入一个或多个衔接子和/或核苷酸标签和/或条形码核苷酸序列到一个或典型地多个靶核苷酸序列的方法。在具体实施方案中，产生具有衔接子的核酸片段，例如，适合用在高通量DNA测序中的核酸片段。在其他实施方案中，关于反应混合物的信息被编码在反应产物中。例如，如果核酸扩增在分别的反应体积中进行，可以期望回收内含物用于后续分析，例如，通过PCR和/或核酸测序。分别的反应体积的内含物可分别分析并将结果与最初的反应体积关联。可选地，颗粒/反应体积身份可被编码在反应产物中，例如，如以下有关多引物核酸扩增方法讨论的。而且，这两种策略可组合从而编码分别的反应体积的组，从而该组中的每个反应体积是独特地可辨识的，并随后汇集，然后分别地分析每个池。For a number of applications, it is necessary or desirable to incorporate nucleic acid sequences into target nucleic acids, for example, derived from samples such as biological samples. In certain embodiments, the incorporated sequences can aid in further analysis of the target nucleic acids. Therefore, methods are described herein that can be used to incorporate one or more adapters and/or nucleotide tags and/or barcode nucleotide sequences into one or typically multiple target nucleotide sequences. In specific embodiments, nucleic acid fragments having adapters are produced, for example, nucleic acid fragments suitable for use in high-throughput DNA sequencing. In other embodiments, information about the reaction mixture is encoded in the reaction products. For example, if nucleic acid amplification is performed in separate reaction volumes, it is desirable to recover the contents for subsequent analysis, for example, by PCR and/or nucleic acid sequencing. The contents of the separate reaction volumes can be analyzed separately and the results associated with the initial reaction volumes. Alternatively, the particle/reaction volume identity can be encoded in the reaction products, for example, as discussed below for multi-primer nucleic acid amplification methods. Moreover, these two strategies can be combined to encode groups of separate reaction volumes so that each reaction volume in the group is uniquely identifiable and subsequently pooled and each pool analyzed separately.

在某些实施方案中，本发明提供扩增方法，其中条形码核苷酸序列和帮助DNA测序的另外的核苷酸序列被加入靶核苷酸序列。条形码核苷酸序列可以编码信息，例如像样品来源、它附加(attached)于其上的相关的靶核酸序列。加入的序列可例如，用作DNA测序引物的结合位点。条形码化靶核苷酸序列可以增加在一个单一测定中针对一个或多个靶可进行分析的样品的数量，同时将测定成本的增加降至最低。这些方法特别适合用于增加微流体装置上进行测定的效率。In certain embodiments, the present invention provides amplification methods in which barcode nucleotide sequences and additional nucleotide sequences that assist in DNA sequencing are added to a target nucleotide sequence. The barcode nucleotide sequence can encode information such as the source of the sample, the associated target nucleic acid sequence to which it is attached. The added sequence can, for example, be used as a binding site for a DNA sequencing primer. The barcoded target nucleotide sequence can increase the number of samples that can be analyzed for one or more targets in a single assay while minimizing the increase in assay costs. These methods are particularly suitable for increasing the efficiency of assays performed on microfluidic devices.

定义definition

除非另外说明，权利要求书以及说明书中所使用的术语是如下面列出定义的。为了清楚的目的这些术语被专门地定义，但是所有这些定义都与本领域技术人员如何理解这些术语的情况一致。Unless otherwise stated, the terms used in the claims and specification are defined as listed below. These terms are specifically defined for the purposes of clarity, but all definitions are consistent with how those skilled in the art understand these terms.

术语“邻近(adjacent)”，当在此使用指核酸中的两个核苷酸序列时，可以指核苷酸序列间隔0至约20个核苷酸、更具体地说，在约1至约10个核苷酸的范围内，或直接地彼此邻接(abut)的序列。如本领域技术人员领会的，将连接在一起的两条核苷酸序列通常将直接地彼此邻接。The term "adjacent", when used herein to refer to two nucleotide sequences in a nucleic acid, can refer to nucleotide sequences that are separated by 0 to about 20 nucleotides, more specifically, within a range of about 1 to about 10 nucleotides, or sequences that are directly abutting each other. As will be appreciated by those skilled in the art, two nucleotide sequences that are to be joined together will typically be directly abutting each other.

术语“核酸”是指核苷酸聚合物，并且除非另外限制，包括天然核苷酸的已知的类似物，这些类似物能够以与天然存在的核苷酸类似的方式起作用(例如杂交)。The term "nucleic acid" refers to a polymer of nucleotides and, unless otherwise limited, includes known analogs of natural nucleotides that can function (eg, hybridize) in a manner similar to naturally occurring nucleotides.

术语核酸包括任何形式的DNA或RNA，包括例如基因组DNA；互补DNA(cDNA)，互补DNA是mRNA的DNA表示，通常通过信使RNA(mRNA)的逆转录、或者通过扩增而得到；合成方式或通过扩增产生的DNA分子；以及mRNA。The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA), or by amplification; DNA molecules produced synthetically or by amplification; and mRNA.

术语核酸包括双链或三链核酸，以及单链分子。在双链或三链核酸中，核酸链不必共同延伸(即双链核酸不必沿着两个链的整个长度是双链的)。The term nucleic acid includes double-stranded or triple-stranded nucleic acids, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acids, the nucleic acid strands are not necessarily coextensive (ie, a double-stranded nucleic acid is not necessarily double-stranded along the entire length of both strands).

不是沿着两条链的完整长度为双链的双链核酸具有在本文称为"粘末端"或"尾序列"的5'或3'延伸(extension)。术语"粘末端"通常用于指相对短的5'或3'延伸，诸如由限制性酶产生的，而术语"尾序列"通常用于指更长的5'或3'延伸。Double-stranded nucleic acids that are not double-stranded along the entire length of both strands have 5' or 3' extensions referred to herein as "sticky ends" or "tail sequences." The term "sticky ends" is generally used to refer to relatively short 5' or 3' extensions, such as those produced by restriction enzymes, while the term "tail sequence" is generally used to refer to longer 5' or 3' extensions.

本文使用的术语"简并序列"表示多个分子中的序列，其中存在多个不同的核苷酸序列。例如，可以存在简并序列的所有可能序列。As used herein, the term "degenerate sequence" refers to a sequence in a plurality of molecules in which a plurality of different nucleotide sequences exist. For example, all possible sequences of a degenerate sequence may exist.

术语"简并尾序列"用于描述多个分子中的尾序列，其中尾序列具有多个不同的核苷酸序列；例如，多个分子中可以存在所有可能的不同的核苷酸序列(每个尾1个)。The term "degenerate tail sequence" is used to describe a tail sequence in multiple molecules, where the tail sequence has multiple different nucleotide sequences; for example, all possible different nucleotide sequences (1 per tail) can be present in the multiple molecules.

术语核酸还包括它们的任何化学修饰，例如通过甲基化和/或通过加帽。核酸修饰可以包括加入化学基团，这些化学基团将额外的电荷、可极化性、氢键、静电作用、以及官能性结合到单独的核酸碱基上或结合到作为整体的核酸上。这类修饰可以包括碱基修饰，例如2'-位置糖修饰、5-位置嘧啶修饰、8-位置嘌呤修饰、在胞嘧啶环外胺上的修饰、5-溴-尿嘧啶的取代、主链修饰、稀有碱基配对组合，例如异碱基异胞苷以及异鸟嘌呤等。The term nucleic acid also includes any chemical modification thereof, for example, by methylation and/or by capping. Nucleic acid modifications can include the addition of chemical groups that are bonded to the individual nucleic acid bases or to the overall nucleic acid with extra charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality. This type of modification can include base modifications, for example 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, substitutions of modifications on the amine outside the cytosine ring, 5-bromo-uracil, main chain modifications, rare base pairing combinations, for example isobases isocytidine and isoguanine etc.

更具体地说，在某些实施方案中，核酸可以包括多聚脱氧核糖核苷酸类(包含2-脱氧-D-核糖)、多聚核糖核苷酸类(包含D-核糖)、以及任何其他类型的核酸，该核酸是嘌呤或嘧啶碱的N-或C-糖苷、以及其他包含非核苷酸主链的聚合物，例如，聚酰胺(例如，核酸肽类(PNA))以及聚吗啉代(可从Anti-Virals,Inc.,Corvallis,Oregon以Neugene商购)聚合物、以及其他合成的序列特异性核酸聚合物，其条件是这些聚合物包含处于允许碱基配对以及碱基堆集的构型的核碱基，例如在DNA以及RNA中发现的碱基配对以及碱基堆集一样。术语核酸还包括锁核酸类(LNA)，在美国专利号6,794,499、6,670,461、6,262,490和6,770,748中对这些锁核酸进行了说明，就它们的LNA的披露而言，这些专利通过引用以其全部内容并入本文。More specifically, in certain embodiments, nucleic acids can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids (PNA)) and polymorpholino (commercially available as Neugene from Anti-Virals, Inc., Corvallis, Oregon) polymers, and other synthetic sequence-specific nucleic acid polymers, provided that these polymers contain nucleobases in a configuration that permits base pairing and base stacking, such as those found in DNA and RNA. The term nucleic acid also includes locked nucleic acids (LNAs), which are described in US Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are incorporated herein by reference in their entireties for their disclosure of LNAs.

可以从完全化学合成方法例如固相介导的化学合成，从生物来源例如通过从产生核酸的任何物种中分离，或从包括通过分子生物学工具例如DNA复制、PCR扩增、逆转录处理核酸的方法，或从这些方法的组合中得到核酸。Nucleic acids can be obtained from completely chemical synthesis methods, such as solid phase mediated chemical synthesis, from biological sources, such as by isolation from any species that produces nucleic acids, or from methods that involve manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of these methods.

核酸分子中元件的顺序在本文通常描述为5'至3'。在双链分子的情形中，根据约定，"上"链通常从5'至3'显示，元件的顺序在本文参考上链描述。The order of elements in a nucleic acid molecule is generally described herein as 5' to 3'. In the case of a double-stranded molecule, the "top" strand is generally shown from 5' to 3' by convention, and the order of elements is described herein with reference to the top strand.

在此使用的术语“靶核酸”是指在本发明的方法中有待检测的特定核酸。As used herein, the term "target nucleic acid" refers to a specific nucleic acid to be detected in the methods of the present invention.

如在此使用的术语“靶核苷酸序列”是指包括靶核酸的核苷酸序列的分子，像，例如，通过扩增靶核酸而得到的扩增产物或当将RNA靶核酸逆转录时所生成的cDNA。The term "target nucleotide sequence" as used herein refers to a molecule including the nucleotide sequence of a target nucleic acid, such as, for example, an amplification product obtained by amplifying a target nucleic acid or a cDNA generated when an RNA target nucleic acid is reverse transcribed.

如在此使用的术语“互补”是指在两个核苷酸之间精确配对的能力。即，如果在一个给定位置上一个核酸的核苷酸能够与另一个核酸的核苷酸形成氢键，则这两个核酸被认为在该位置上是彼此互补的。两个单链核酸分子之间的互补性可以是“部分的”，其中仅一些核苷酸进行结合，或当单链分子之间存在完全互补性时，它可以是完全的。核酸链之间互补性的程度对于核酸分子之间杂交的效率以及强度具有显著影响。如果第一核苷酸序列与第二核苷酸序列互补，则第一核苷酸序列被称为是第二序列的"互补物"。如果第一核苷酸序列与第二序列的反向(即，核苷酸的顺序是反向的)的序列互补，则第一核苷酸序列被称为是第二序列的"反向互补物"。As used herein, the term "complementarity" refers to the ability to accurately match between two nucleotides. That is, if the nucleotides of a nucleic acid can form hydrogen bonds with the nucleotides of another nucleic acid at a given position, the two nucleic acids are considered to be complementary to each other at that position. The complementarity between two single-stranded nucleic acid molecules can be "partial," with only some nucleotides combining, or when there is complete complementarity between the single-stranded molecules, it can be complete. The degree of complementarity between the nucleic acid chains has a significant impact on the efficiency and intensity of hybridization between the nucleic acid molecules. If the first nucleotide sequence is complementary to the second nucleotide sequence, the first nucleotide sequence is referred to as the "complement" of the second sequence. If the first nucleotide sequence is complementary to the reverse (that is, the order of nucleotides is reverse) sequence of the second sequence, the first nucleotide sequence is referred to as the "reverse complement" of the second sequence.

“特异性杂交”是指在定义的严格条件下核酸结合到靶核苷酸序列上，不实质性地结合到杂交混合物中存在的其他核苷酸序列上。本领域普通技术人员应当知道放松杂交条件的严格性将允许容忍序列错配。"Specific hybridization" refers to the binding of a nucleic acid to a target nucleotide sequence under defined stringent conditions without substantially binding to other nucleotide sequences present in the hybridization mixture. One of ordinary skill in the art will appreciate that relaxing the stringency of hybridization conditions will allow for tolerance of sequence mismatches.

在具体实施方案中，在严格杂交条件下进行杂交。短语“严格杂交条件”总体上是指在定义的离子强度和pH下对于特定序列而言温度在低于解链温度(T_m)从约5℃至约20℃或25℃的范围内。如在此使用的，T_m是双链核酸分子的群体被半解离成单链的温度。用于计算核酸的T_m的方法在本领域中是熟知的(参见，例如，Berger和Kimmel(1987)METHODS INENZYMOLOGY,VOL.152:GUIDE TO MOLECULAR CLONING TECHNIQUES,San Diego:AcademicPress,Inc.与Sambrook等人.(1989)MOLECULAR CLONING:A LABORATORY MANUAL,第2版,VOLS.1-3,Cold Spring Harbor Laboratory)，两者都通过引用并入本文)。如标准参考文献所指出的，当核酸处于1M NaCl的水溶液中时，可以通过下面等式计算T_m值的简单估计T_m＝81.5+0.41(％G+C)(参见，例如，Anderson和Young,Quantitative FilterHybridization in NUCLEIC ACID HYBRIDIZATION(1985))。杂交体的解链温度(以及因此用于严格杂交的条件)被多种因素影响，例如引物或探针的长度以及性质(DNA、RNA、碱基组成)以及靶核酸的性质(DNA、RNA、碱基组成、存在于溶液中或固定的等)，以及盐和其他组分的浓度(例如，甲酰胺、硫酸葡聚糖、聚乙二醇的存在与否)。这些因素的影响是熟知的并且在本领域的标准参考文献中进行了讨论。适合用于实现大多数序列的特异性杂交的示例性的严格条件是：在pH7至少约60℃的温度以及约0.2摩尔/升的盐浓度。In a specific embodiment, hybridization is carried out under stringent hybridization conditions.Phrase " stringent hybridization conditions " generally refers to that the temperature is lower than the melting temperature (_Tm ) in the range of about 5 DEG C to about 20 DEG C or 25 DEG C for a particular sequence under defined ionic strength and pH.As used herein,_Tm is the temperature at which the colony of double-stranded nucleic acid molecules is half-dissociated into single strands.The method for calculating the_Tm of nucleic acid is well known in the art (see, e.g., Berger and Kimmel (1987) METHODS INENZYMOLOGY, VOL.152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego:Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition, VOLS.1-3, Cold Spring Harbor Laboratory), both of which are incorporated herein by reference). As indicated in standard reference texts, a simple estimate of the_Tm value when the nucleic acid is in 1 M NaCl in water can be calculated by the equation_Tm = 81.5 + 0.41 (% G + C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of the hybrid (and therefore the conditions for stringent hybridization) is affected by a variety of factors, such as the length and nature of the primer or probe (DNA, RNA, base composition) and the nature of the target nucleic acid (DNA, RNA, base composition, in solution or immobilized, etc.), as well as the concentrations of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and discussed in standard reference texts in the art. Exemplary stringent conditions suitable for achieving specific hybridization of most sequences are a temperature of at least about 60°C at pH 7 and a salt concentration of about 0.2 mol/L.

使用术语“寡核苷酸”是指相对短的核酸，该核酸总体上短于200个核苷酸、更特别地短于100个核苷酸、最特别地短于50个核苷酸。典型地，寡核苷酸是单链DNA分子。The term "oligonucleotide" is used to refer to a relatively short nucleic acid, generally shorter than 200 nucleotides, more particularly shorter than 100 nucleotides, most particularly shorter than 50 nucleotides. Typically, an oligonucleotide is a single-stranded DNA molecule.

术语"衔接子"用于指在使用中变得附加到核酸的一个或两个末端的核酸。衔接子可以是单链、双链的，或可包含单链和双链部分。The term "adapter" is used to refer to a nucleic acid that, in use, becomes attached to one or both ends of a nucleic acid. An adaptor can be single-stranded, double-stranded, or can contain single-stranded and double-stranded portions.

术语“引物”是指在适宜的条件下(即在四种不同核苷三磷酸以及聚合反应试剂例如DNA或RNA聚合酶或逆转录酶的存在下)在适宜的缓冲液中并且在适宜的温度下能够与核酸杂交(也称为“退火”)并且用作核苷酸(RNA或DNA)聚合反应的起始位点的寡核苷酸。引物的适当的长度取决于引物的预期用途，但是典型地引物是至少7个核苷酸长度，更典型地范围从10个核苷酸至30个核苷酸，或甚至更典型地从15个核苷酸至30个核苷酸长度。其他引物可以是稍微更长的，例如30至50个核苷酸长度。在此上下文中，“引物长度”是指杂交到互补的“靶”序列上并且引发核苷酸合成的寡核苷酸或核酸的部分。短引物分子总体上需要更冷的温度以与模板形成足够稳定的杂交复合体。引物不必反映模板的确切序列但是必须是足够互补的以与模板杂交。术语“引物位点”或“引物结合位点”是指引物杂交到其上的靶核酸的区段。The term "primer" refers to an oligonucleotide that can hybridize to nucleic acid (also referred to as "annealing") and serve as a starting site for nucleotide (RNA or DNA) polymerization reaction under suitable conditions (i.e., in the presence of four different nucleoside triphosphates and polymerization reagents such as DNA or RNA polymerase or reverse transcriptase) in a suitable buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but typically a primer is at least 7 nucleotides in length, more typically ranging from 10 nucleotides to 30 nucleotides, or even more typically from 15 nucleotides to 30 nucleotides in length. Other primers can be slightly longer, such as 30 to 50 nucleotides in length. In this context, "primer length" refers to the oligonucleotide or nucleic acid portion that hybridizes to a complementary "target" sequence and initiates nucleotide synthesis. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with templates. Primers do not necessarily reflect the exact sequence of the template but must be sufficiently complementary to hybridize with the template. The term "primer site" or "primer binding site" refers to the segment of the target nucleic acid to which the primer hybridizes.

如果引物、或它的一部分杂交到另一个核酸中的核苷酸序列上，引物被说成退火到该核酸上。引物杂交到特定核苷酸序列上的陈述不旨在暗示该引物完全地或唯一地杂交到该核苷酸序列上。例如，在某些实施方案中，在此使用的扩增引物被说成“退火到核苷酸标签上”。这种说明包括完全退火到核苷酸标签上的引物以及部分地退火到核苷酸标签上以及部分地退火到邻近核苷酸序列例如靶核苷酸序列)上的引物。这类杂交引物可以增加扩增反应的特异性。If a primer, or a portion thereof, hybridizes to a nucleotide sequence in another nucleic acid, a primer is said to anneal to the nucleic acid. The statement that a primer hybridizes to a specific nucleotide sequence is not intended to imply that the primer hybridizes completely or exclusively to that nucleotide sequence. For example, in certain embodiments, the amplification primers used herein are said to "anneal to a nucleotide tag." This description includes primers that anneal completely to a nucleotide tag and primers that anneal partially to a nucleotide tag and partially to an adjacent nucleotide sequence (e.g., a target nucleotide sequence). Such hybridization primers can increase the specificity of the amplification reaction.

如在此使用的，选择引物“以便避免实质性地退火到靶核酸上”是指选择引物以使得在扩增之后检出的大多数扩增子在下面意义上是“全长的”，即它们是从在靶核酸的每个末端上在预期的位点处引发而得到的，与从靶核酸内引发而得到的扩增子不同，后者生成比预期更短的扩增子。在不同实施方案中，选择引物从而至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、或至少99％是全长的。As used herein, selecting primers "so as to avoid substantial annealing to the target nucleic acid" means selecting primers so that the majority of amplicons detected after amplification are "full-length" in the sense that they result from priming at the expected sites on each end of the target nucleic acid, as opposed to amplicons resulting from priming within the target nucleic acid, which produce shorter amplicons than expected. In various embodiments, primers are selected so that at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are full-length.

术语“引物对”是指一组引物，这组引物包括与有待扩增的DNA序列的5’末端的互补物杂交的5'“上游引物”或“正向引物”，以及与有待扩增的序列的3’末端杂交的3'“下游引物”或“反向引物”。如本领域普通技术人员应当了解的，在具体实施方案中术语“上游”和“下游”或“正向”和“反向”不旨在进行限制，而是提供示意性的方向。The term "primer pair" refers to a set of primers comprising a 5' "upstream primer" or "forward primer" that hybridizes to the complement of the 5' end of the DNA sequence to be amplified, and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. As will be appreciated by those of ordinary skill in the art, in specific embodiments the terms "upstream" and "downstream" or "forward" and "reverse" are not intended to be limiting, but rather to provide illustrative directions.

在使用两个引物对的实施方案中，例如，在扩增反应中，引物对可标为"内侧"和"外侧"引物对以指示其相对位置；即，"内侧"引物被掺入到反应产物(例如，扩增子)中外侧引物被掺入的位置之间的位置。In embodiments where two primer pairs are used, e.g., in an amplification reaction, the primer pairs can be labeled "inside" and "outside" primer pairs to indicate their relative positions; i.e., the "inside" primer is incorporated into the reaction product (e.g., an amplicon) at a position between the positions where the outer primer is incorporated.

在使用三个引物对的实施方案中，例如，在扩增反应中，术语"填充引物"可用于指具有在内侧和外侧引物之间的位置的引物；即，"填充"引物被掺入到反应产物(例如，扩增子)中内侧和外侧引物之间的中间位置。In embodiments where three primer pairs are used, e.g., in an amplification reaction, the term "filler primer" may be used to refer to a primer having a position between the inner and outer primers; that is, the "filler" primer is incorporated into the reaction product (e.g., an amplicon) at a position intermediate between the inner and outer primers.

如果一个引物对可以被用于特定地产生(例如，扩增)给出的反应(例如，扩增)混合物中特定的反应产物(例如，扩增子)，则所述引物对被称为是“独特的”。A primer pair is said to be "unique" if it can be used to specifically produce (eg, amplify) a specific reaction product (eg, amplicon) in a given reaction (eg, amplification) mixture.

“探针”是能够通过一个或多个类型的化学键、通常通过互补碱基配对、经常通过氢键形成结合到互补序列的靶核酸上、因此形成双链结构的核酸。探针结合或杂交到“探针结合位点”上。探针可以用可检出的标记物进行标记以允许容易地检出探针，特别是一旦探针杂交到它的互补靶上时。然而，作为替代方案，探针可以是未标记的，但可以是通过与直接地或间接地标记的配体特异性结合而可以检出的。在尺寸方面探针可以显著地改变。总体上，探针是至少7至15个核苷酸长度。其他的探针是至少20、30或40个核苷酸长度。而其他探针是稍微更长的，为至少50、60、70、80或90个核苷酸长度。而其他探针仍然是更长的，并且是至少100、150、200或更多的核苷酸长度。探针还可以是在由上述值中任何一项所限定的任何范围内的任何长度(例如，15至20个核苷酸长度)。"Probe" is a nucleic acid that can be bound to a target nucleic acid of a complementary sequence by one or more types of chemical bonds, usually by complementary base pairing, often by hydrogen bond formation, thus forming a double-stranded structure. The probe binds to or hybridizes to a "probe binding site". The probe can be labeled with a detectable marker to allow easy detection of the probe, particularly once the probe hybridizes to its complementary target. However, as an alternative, the probe can be unlabeled, but can be detectable by specifically binding to a ligand directly or indirectly labeled. The probe can vary significantly in size. Generally, the probe is at least 7 to 15 nucleotides in length. Other probes are at least 20, 30, or 40 nucleotides in length. Other probes are slightly longer, being at least 50, 60, 70, 80, or 90 nucleotides in length. Other probes are still longer, and are at least 100, 150, 200, or more nucleotides in length. The probe can also be any length (e.g., 15 to 20 nucleotides in length) within any range defined by any one of the above values.

引物或探针可以是与靶核酸序列完全互补的或可以是小于完全互补的。在某些实施方案中，在至少7个核苷酸的序列上，更典型地在10至30个核苷酸范围内的序列上，并且通常在至少14-25个核苷酸的序列上，引物与靶核酸序列的互补物具有至少65％同一性，并且更经常地具有至少75％同一性、至少85％同一性、至少90％同一性、或至少95％、96％、97％、98％或99％同一性。应当理解的是总体上令人希望的是某些碱基(例如引物的3’碱基)是与靶核酸序列的相应的碱基完全互补的。在严格杂交条件下引物和探针典型地退火到靶序列上。Primer or probe can be with target nucleic acid sequence complete complementarity or can be less than complete complementarity.In certain embodiments, on the sequence of at least 7 nucleotides, more typically on the sequence in the range of 10 to 30 nucleotides, and conventionally on the sequence of at least 14-25 nucleotides, primer and the complement of target nucleic acid sequence have at least 65% homogeneity, and more frequently have at least 75% homogeneity, at least 85% homogeneity, at least 90% homogeneity or at least 95%, 96%, 97%, 98% or 99% homogeneity.It should be understood that what is desirable generally is that some base (for example 3 ' base of primer) is with the corresponding base complete complementarity of target nucleic acid sequence.Under stringent hybridization conditions, primer and probe are typically annealed on the target sequence.

在此使用术语“核苷酸标签”是指被加入到靶核苷酸序列中的预定的核苷酸序列。核苷酸标签可以编码关于该靶核苷酸序列的一项信息，例如靶核苷酸序列的身份或从中得到靶核苷酸序列的样品的身份。在某些实施方案中，这类信息可以被编码到一个或多个核苷酸标签中，例如两个核苷酸标签的组合，在靶核苷酸序列的两个末端之一上的一个可以编码靶核苷酸序列的身份。As used herein, the term "nucleotide tag" refers to a predetermined nucleotide sequence that is added to a target nucleotide sequence. The nucleotide tag can encode information about the target nucleotide sequence, such as the identity of the target nucleotide sequence or the identity of the sample from which the target nucleotide sequence was obtained. In certain embodiments, such information can be encoded in one or more nucleotide tags, for example, a combination of two nucleotide tags, one at each end of the target nucleotide sequence, that can encode the identity of the target nucleotide sequence.

本文使用的术语"亲和性标签"是指分子被结合伴侣特异性结合的部分。该部分可以，但不需要是，核苷酸序列。特异性结合可用于帮助加亲和性标签的分子的亲和性纯化。As used herein, the term "affinity tag" refers to a portion of a molecule that is specifically bound by a binding partner. This portion can, but need not be, a nucleotide sequence. Specific binding can be used to facilitate affinity purification of the affinity-tagged molecule.

术语"转座子末端"是指能够被转座酶附加到核酸的寡核苷酸。The term "transposon end" refers to an oligonucleotide that can be appended to a nucleic acid by a transposase.

如在此使用的术语“条形码引物”是指包括特异性条形码核苷酸序列的引物，该核苷酸序列编码了关于当条形码引物被用于扩增反应时所生成的扩增子的信息。例如，可以使用不同的条形码引物以从大量不同样品的每一个中扩增一个或多个靶序列，这样使得条形码核苷酸序列指示得到的扩增子的样品来源。As used herein, the term "barcode primer" refers to a primer that includes a specific barcode nucleotide sequence that encodes information about the amplicon generated when the barcode primer is used in an amplification reaction. For example, different barcode primers can be used to amplify one or more target sequences from each of a plurality of different samples, such that the barcode nucleotide sequence indicates the sample origin of the resulting amplicon.

如在此使用的，术语“编码反应”是指其中至少一个核苷酸标签被加入到靶核苷酸序列中的反应。例如可以通过“编码PCR”加入核苷酸标签，其中至少一个引物包括靶特异性部分以及位于该靶特异性部分的5’末端上的核苷酸标签，以及第二引物，该第二引物包括仅靶特异性部分或靶特异性部分和位于该靶特异性部分的5’末端上的核苷酸标签。关于可用于编码PCR的PCR方案的示意性实例，参见未决的WO申请US03/37808以及美国专利号6,605,451。还可以通过“编码连接”反应来加入核苷酸标签，该反应可以包括连接反应，其中至少一个引物包括靶特异性部分以及位于该靶特异性部分的5’末端上的核苷酸标签，以及第二引物，该第二引物包括仅靶特异性部分或靶特异性部分和位于该靶特异性部分的5'末端上的核苷酸标签。示意性的编码连接反应被描述于例如美国专利公布号2005/0260640中，该专利通过引用以其全部内容并入本文，并且特别地是针对连接反应。As used herein, the term "encoding reaction" refers to a reaction in which at least one nucleotide tag is added to a target nucleotide sequence. For example, the nucleotide tag can be added by "encoding PCR", wherein at least one primer includes a target-specific portion and a nucleotide tag located at the 5' end of the target-specific portion, and a second primer that includes only the target-specific portion or the target-specific portion and a nucleotide tag located at the 5' end of the target-specific portion. For illustrative examples of PCR protocols that can be used for encoding PCR, see pending WO application US03/37808 and U.S. Patent No. 6,605,451. The nucleotide tag can also be added by a "encoding ligation" reaction, which can include a ligation reaction, wherein at least one primer includes a target-specific portion and a nucleotide tag located at the 5' end of the target-specific portion, and a second primer that includes only the target-specific portion or the target-specific portion and a nucleotide tag located at the 5' end of the target-specific portion. Schematic encoding ligation reactions are described, for example, in US Patent Publication No. 2005/0260640, which is incorporated herein by reference in its entirety, and particularly with respect to ligation reactions.

如在此所使用的“编码反应”可生成“加标签的靶核苷酸序列”，该核苷酸序列包括连接到靶核苷酸序列上的核苷酸标签。As used herein, an "encoding reaction" can generate a "tagged target nucleotide sequence" that includes a nucleotide tag attached to the target nucleotide sequence.

如在此使用的，提及引物的部分，术语“靶特异性”核苷酸序列是指在适当的退火条件下能够特异性退火到靶核酸或靶核苷酸序列上的序列。As used herein, in reference to a portion of a primer, the term "target-specific" nucleotide sequence refers to a sequence that is capable of specifically annealing to a target nucleic acid or target nucleotide sequence under appropriate annealing conditions.

如在此使用的，提及引物的部分，术语“核苷酸标签特异性核苷酸序列”是指在适当的退火条件下能够特异性地退火到核苷酸标签上的序列。As used herein, with reference to a portion of a primer, the term "nucleotide tag-specific nucleotide sequence" refers to a sequence that can specifically anneal to a nucleotide tag under appropriate annealing conditions.

根据本发明的教导的扩增包括使至少一个靶核酸的至少一部分再生的任何手段，典型地是以模板依赖性方式，包括但不限于，用于线性或指数扩增核酸序列的广泛的技术。用于完成扩增步骤的示意性手段包括连接酶链式反应(LCR)、连接酶检测反应(LDR)、连接紧接着Q-复制酶扩增、PCR、引物延伸、链置换扩增(SDA)、超支化链置换扩增、多重置换扩增(MDA)、基于核酸链的扩增(NASBA)、两步多重扩增、滚环扩增(RCA)、等，包括它们的多种形式以及组合，例如但不限于OLA/PCR、PCR/OLA、LDR/PCR、PCR/PCR/LDR、PCR/LDR、LCR/PCR、PCR/LCR(也称为组合链反应-CCR)，等。这类技术的说明可以在以下及其他来源中找到：Ausbel等人；PCR Primer:A Laboratory Manual,Diffenbach编著,Cold Spring HarborPress(1995)；The Electronic Protocol Book,Chang Bioscience(2002)；Msuih等人,J.Clin.Micro.34:501-07(1996)；The Nucleic Acid Protocols Handbook,R.Rapley编著,Humana Press,Totowa,N.J.(2002)；Abramson等人,Curr Opin Biotechnol.1993Feb.；4(1):41-7,美国专利号6,027,998；美国专利号6,605,451,Barany等人,PCT公布号WO97/31256；Wenz等人,PCT公布号WO 01/92579；Day等人,Genomics,29(1):152-162(1995),Ehrlich等人,Science 252:1643-50(1991)；Innis等人,PCR Protocols:A Guide toMethods and Applications,Academic Press(1990)；Favis等人,NatureBiotechnology18:561-64(2000)；和Rabenau等人,Infection 28:97-102(2000)；Belgrader,Barany和Lubin,Development of a Multiplex Ligation DetectionReaction DNA Typing Assay,Sixth International Symposium on HumanIdentification,1995(在下面的互联网址上可得：promega.com/geneticidproc/ussymp6proc/blegrad.html-)；LCR Kit Instruction Manual,Cat.#200520,Rev.#050002,Stratagene,2002；Barany,Proc.Natl.Acad.Sci.USA 88:188-93(1991)；Bi和Sambrook,Nucl.Acids Res.25:2924-2951(1997)；Zirvi等人,Nucl.Acid Res.27:e40i-viii(1999)；Dean等人,Proc Natl Acad Sci USA 99:5261-66(2002)；Barany和Gelfand,Gene109:1-11(1991)；Walker等人,Nucl.Acid Res.20:1691-96(1992)；Polstra等人,BMCInf.Dis.2:18-(2002)；Lage等人,Genome Res.2003Feb.；13(2):294-307,和Landegren等人,Science 241:1077-80(1988),Demidov,V.,Expert Rev Mol Diagn.2002 Nov.；2(6):542-8.,Cook等人,J Microbiol Methods.2003 May；53(2):165-74,Schweitzer等人,CurrOpin Biotechnol.2001 Feb.；12(l):21-7,美国专利号5,830,711,美国专利号6,027,889,美国专利号5,686,243,PCT公布号WO0056927A3,和PCT公布号WO9803673A1。Amplification according to the teachings of the present invention includes any means of regenerating at least a portion of at least one target nucleic acid, typically in a template-dependent manner, including but not limited to, a wide range of techniques for linear or exponential amplification of nucleic acid sequences. Schematic means for completing the amplification step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid chain-based amplification (NASBA), two-step multiple amplification, rolling circle amplification (RCA), etc., including their various forms and combinations, such as but not limited to OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also referred to as combined chain reaction-CCR), etc. Descriptions of such techniques can be found in, among other sources: Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.; 4(1):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1):152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the internet at: promega.com/geneticidproc/ussymp6proc/blegrad.html-); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acids Res. 20: 1691-96 (1992); Polstra et al., BMC Inf. Dis. 2: 18- (2002); Lage et al., Genome Res. 2003 Feb.; 13(2): 294-307, and Landegren et al., Science 241: 1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov.; 2(6): 542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 Feb.; 12(l):21-7, U.S. Patent No. 5,830,711, U.S. Patent No. 6,027,889, U.S. Patent No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

在一些实施方案中，扩增包括下面各项的连续步骤的至少一个循环：将至少一个引物与至少一个靶核酸中互补的或基本上互补的序列一起退火；使用聚合酶以模板依赖性方式合成核苷酸的至少一个链；并且将新形成的核酸双链变性以分离这些链。该循环可以被重复或可以不被重复。扩增可以包括热循环或能以等温方式完成。In some embodiments, amplification includes at least one cycle of the following sequential steps: annealing at least one primer to a complementary or substantially complementary sequence in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification may include thermal cycling or may be performed isothermally.

在此使用术语“qPCR”是指定量实时聚合酶链式反应(PCR)，该反应还被称为“实时PCR”或“动力学聚合酶链式反应”。The term "qPCR" is used herein to refer to quantitative real-time polymerase chain reaction (PCR), which is also known as "real-time PCR" or "kinetic polymerase chain reaction."

本文提及参数使用的术语"大致上"表示，该参数足以提供有用的结果。因此，"大致上互补"在应用于核酸序列时通常表示足够互补以在所描述的情景中起作用。通常，大致上互补表示足够互补以在采用的条件下杂交。在本文描述的一些实施方案中，反应产物必须区分于未反应的引物。在这种情形中，"反应产物是大致上双链的"的陈述和"引物是大致上单链的"的陈述表示，双链反应产物和单链引物的量之间存在足够的差异，从而可确定反应产物的存在和/或量。The term "substantially" used herein in reference to a parameter means that the parameter is sufficient to provide a useful result. Thus, "substantially complementary" when applied to nucleic acid sequences generally means sufficiently complementary to function in the described scenario. Typically, substantially complementary means sufficiently complementary to hybridize under the conditions employed. In some embodiments described herein, the reaction product must be distinguished from unreacted primers. In this case, the statement that "the reaction product is substantially double-stranded" and the statement that "the primer is substantially single-stranded" indicate that there is sufficient difference between the amount of the double-stranded reaction product and the amount of the single-stranded primer to determine the presence and/or amount of the reaction product.

“试剂”广义地是指除了分析物(例如被分析的核酸)之外用于反应中的任何剂。用于核酸扩增反应的示意性试剂包括但不限于，缓冲剂、金属离子、聚合酶、逆转录酶、引物、模板核酸、核苷酸、标记物、染料、核酸酶、等。用于酶反应的试剂包括例如底物、辅因子、缓冲剂、金属离子、抑制剂、以及活化剂。"Reagent" broadly refers to any agent used in a reaction other than the analyte (e.g., the nucleic acid being analyzed). Illustrative reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, and the like. Reagents for enzymatic reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

在此使用术语“通用检测探针”是指鉴别扩增产物存在的任何探针，无论产物中存在的靶核苷酸序列的身份如何。The term "universal detection probe" is used herein to refer to any probe that identifies the presence of an amplification product regardless of the identity of the target nucleotide sequence present in the product.

在此使用术语“通用qPCR探针”是指在qPCR期间鉴别扩增产物的存在的任何这类探针。在具体实施方案中，根据本发明的核苷酸标签可以包括检测探针例如通用qPCR探针结合到其上的核苷酸序列。当标签被加到靶核苷酸序列的两个末端上时，如果想要的话每个标签可以包括被检测探针识别的序列。这类序列的组合可以编码关于加标签的靶核苷酸序列的身份或样品来源的信息。在其他实施方案中，一个或多个扩增引物可以包括检测探针例如通用qPCR探针结合到其上的核苷酸序列。以这种方式，在本发明的方法的扩增步骤期间一个、两个、或多个探针结合位点可以被加到扩增产物中。本领域普通技术人员知道在预扩增期间(如果执行的话)引入多个探针结合位点的可能性，并且扩增有助于多重检测，其中可以在给定的扩增混合物或它的等份中检出两个或多个不同的扩增产物。The term "universal qPCR probe" is used herein to refer to any such probe that identifies the presence of an amplification product during qPCR. In a specific embodiment, a nucleotide tag according to the present invention may include a nucleotide sequence to which a detection probe, such as a universal qPCR probe, binds. When tags are added to both ends of a target nucleotide sequence, each tag may include a sequence recognized by a detection probe, if desired. The combination of such sequences can encode information about the identity of the tagged target nucleotide sequence or the source of the sample. In other embodiments, one or more amplification primers may include a nucleotide sequence to which a detection probe, such as a universal qPCR probe, binds. In this way, one, two, or more probe binding sites can be added to the amplification product during the amplification step of the method of the present invention. One of ordinary skill in the art is aware of the possibility of introducing multiple probe binding sites during pre-amplification (if performed), and amplification facilitates multiplex detection, where two or more different amplification products can be detected in a given amplification mixture or its aliquots.

术语“通用检测探针”还旨在包括用可检出标记物(例如，荧光标记物)标记的引物、以及非序列特异性探针，例如DNA结合染料，包括双链DNA(dsDNA)染料，例如SYBRGreen。The term "universal detection probe" is also intended to include primers labeled with a detectable label (e.g., a fluorescent label), and non-sequence specific probes, such as DNA binding dyes, including double-stranded DNA (dsDNA) dyes such as SYBR Green.

如在此使用的术语“标记物”是指能够用于提供可检出的和/或可定量的信号的任何原子或分子。具体地，可以将标记物直接地或间接地附加到核酸或蛋白上。可以附加到探针上的适合的标记物包括但不限于放射性同位素、荧光团、生色团、质量标记、电子致密颗粒、磁性颗粒、自旋标记物、发出化学发光的分子、电化学活性分子、酶、辅因子、以及酶底物。As used herein, the term "label" refers to any atom or molecule that can be used to provide a detectable and/or quantifiable signal. Specifically, the label can be attached directly or indirectly to a nucleic acid or protein. Suitable labels that can be attached to the probe include, but are not limited to, radioisotopes, fluorophores, chromophores, mass markers, electron-dense particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

本文使用的术语"染色剂"总体上是指结合反应或检验混合物的组分以帮助检测该组分的任何有机的或无机的分子。As used herein, the term "stain" generally refers to any organic or inorganic molecule that binds to a component of a reaction or assay mixture to aid in detection of that component.

如在此所使用的术语“染料”总体上是指在大于或等于340nm的波长处吸收电磁辐射的任何有机的或无机的分子。The term "dye" as used herein generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation at a wavelength greater than or equal to 340 nm.

如在此使用的术语“荧光染料”总体上是指当通过电磁辐射来源例如灯、光电二极管或激光进行照射时通过荧光机理发出较长波长电磁辐射的任何染料。The term "fluorescent dye" as used herein generally refers to any dye that emits longer wavelength electromagnetic radiation by a fluorescence mechanism when illuminated by an electromagnetic radiation source such as a lamp, photodiode, or laser.

术语“弹性体”具有本领域中所使用的一般性含义。因此，例如，Allcock等人(Contemporary Polymer Chemistry,第2版)说明了弹性体总体上作为聚合物存在于它们的玻璃转化温度与液化温度之间的温度下。弹性材料显示出弹性特性，因为这些聚合物链易于经受扭转移动从而允许使主链响应于力而解螺旋，在缺少该力时主链再螺旋(recoiling)从而呈现出先前的形状。总体上，当施加力时弹性体变形，但是然后当去除该力时，它们返回到它们的初始形状。The term "elastomer" has a general meaning used in this area. Therefore, for example, Allcock et al. (Contemporary Polymer Chemistry, 2nd edition) illustrate that elastomers are generally present as polymers at a temperature between their glass transition temperature and the liquidus temperature. Elastomeric materials show elastic properties because these polymer chains are prone to torsion movement, thereby allowing the main chain to unwind in response to a force, and when the force is lacking, the main chain re-spirals (recoiling) thereby presenting a previous shape. In general, elastomers deform when a force is applied, but then when the force is removed, they return to their initial shape.

本文使用的术语"变化"用于指任何差异。变化可以指个体或群体之间的差异。变化涵盖与普通或正常情形的差异。因此，"拷贝数变化"或"突变"可以指与普通或正常拷贝数或核苷酸序列的差异。"表达水平变化"或"剪接变体"可以指对于特定、细胞或组织、发育阶段、状态等不同于普通或正常表达水平或RNA或蛋白的表达水平或RNA或蛋白。As used herein, the term "variation" is used to refer to any difference. Variation can refer to differences between individuals or populations. Variation encompasses differences from the common or normal situation. Thus, "copy number variation" or "mutation" can refer to a difference from the common or normal copy number or nucleotide sequence. "Expression level variation" or "splice variant" can refer to an expression level or RNA or protein that is different from the common or normal expression level or RNA or protein for a particular, cell or tissue, developmental stage, state, etc.

“多态性标志物”或“多态位点”是在其上出现核苷酸序列趋异的基因座。示例性标志物具有至少两个等位基因，每个以大于1％、并且更典型地大于10％或20％的选择群体的频率出现。多态性位点可以是小至一个碱基对。多态性标志物包括限制性片段长度多态性(RFLP)、可变数量串联重复(VNTR)、超可变区、小卫星序列、双核苷酸重复、三核苷酸重复、四核苷酸重复、简单序列重复、缺失、以及插入元件，例如Alu。首先鉴定的等位基因形式被任意指定为参考形式并且其他等位基因形式被指定为替代的或变体的等位基因。在选择的群体中最频繁出现的等位基因形式有时被称为野生型。对于等位基因形式而言，二倍体生物可以是纯合的或杂合的。双等位基因多态性具有两种形式。三等位基因多态性具有三种形式。"Polymorphic markers" or "polymorphic sites" are loci at which nucleotide sequence divergence occurs. Exemplary markers have at least two alleles, each occurring at a frequency greater than 1%, and more typically greater than 10% or 20%, of the selected population. Polymorphic sites can be as small as one base pair. Polymorphic markers include restriction fragment length polymorphisms (RFLPs), variable number tandem repeats (VNTRs), hypervariable regions, minisatellite sequences, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, deletions, and insertion elements, such as Alu. The allelic form first identified is arbitrarily designated as a reference form and other allelic forms are designated as alternative or variant alleles. The allelic form that most frequently occurs in a selected population is sometimes referred to as wild type. For allelic form, diploid organisms can be homozygous or heterozygous. Biallelic polymorphisms have two forms. Triallelic polymorphisms have three forms.

“单核苷酸多态性”(SNP)出现在被单核苷酸占据的多态性位点，这个位点是等位基因序列之间出现变化的位点。该位点通常在等位基因的高度保守的序列(例如，在群体的小于1/100或1/1000的成员中发生变化的序列)之前或之后。SNP通常由于在多态性位点处一个核苷酸被取代成另一个核苷酸而出现。转变(transition)是一个嘌呤被替换成另一个嘌呤或者一个嘧啶被替换成另一个嘧啶。颠换(transversion)是嘌呤被替换成嘧啶或反之亦然。SNP还可以通过相对于参考等位基因缺失一个核苷酸或插入一个核苷酸而产生。"Single nucleotide polymorphism" (SNP) occurs at a polymorphic site occupied by a single nucleotide, and this site is a site where changes occur between allele sequences. This site is usually before or after the highly conserved sequence of the allele (for example, a sequence that changes in a member less than 1/100 or 1/1000 of a population). SNP usually occurs due to a nucleotide being substituted into another nucleotide at the polymorphic site. A transition is when a purine is replaced into another purine or a pyrimidine is replaced into another pyrimidine. A transversion is when a purine is replaced into a pyrimidine or vice versa. SNP can also be produced by deleting a nucleotide or inserting a nucleotide relative to a reference allele.

如本文关于反应、反应混合物、反应体积等使用的，术语"分别的"是指其中反应与其他反应分离地进行的反应、反应混合物、反应体积等。分别的反应、反应混合物、反应体积等包括在液滴中进行的那些(参见，例如，2007年11月13日授权的Quake等人的美国专利号7,294,503，题为"Microfabricated crossflow devices and methods"，其通过引用全文并入本文，尤其是关于形成和分析液滴的装置和方法的描述；2010年1月28日公布的Link等人的美国专利公布号20100022414，题为"Droplet libraries"，其通过引用全文并入本文，尤其是关于形成和分析液滴的装置和方法的描述；和2011年1月6日公布的Miller等人的美国专利公布号20110000560，题为"Manipulation of Micro fluidic Droplets"，其通过引用全文并入本文，尤其是关于形成和分析液滴的装置和方法的描述)，其可以、但不需要是在乳液中，以及其中反应、反应混合物、反应体积等被机械障碍(mechanical barrier)分隔的那些，例如，分别的容器、微量滴定板的分别的孔、或矩阵型微流体装置(matrix-typemicrofluidic device)的分别的区室。As used herein with respect to reactions, reaction mixtures, reaction volumes, and the like, the term "separate" refers to reactions, reaction mixtures, reaction volumes, and the like in which a reaction is performed separately from other reactions. Respective reactions, reaction mixtures, reaction volumes, etc. include those conducted in droplets (see, e.g., U.S. Patent No. 7,294,503 to Quake et al., issued November 13, 2007, entitled “Microfabricated crossflow devices and methods,” which is incorporated herein by reference in its entirety, particularly with respect to devices and methods for forming and analyzing droplets; U.S. Patent Publication No. 20100022414 to Link et al., issued January 28, 2010, entitled “Droplet libraries,” which is incorporated herein by reference in its entirety, particularly with respect to devices and methods for forming and analyzing droplets; and U.S. Patent Publication No. 20110000560 to Miller et al., issued January 6, 2011, entitled “Manipulation of Microfluidic Droplets,” which is incorporated herein by reference in its entirety, particularly with respect to devices and methods for forming and analyzing droplets), which can, but need not, be in emulsions, and where reactions, reaction mixtures, reaction volumes, etc. are mechanically obstructed. These are those separated by a barrier, for example, separate containers, separate wells of a microtiter plate, or separate compartments of a matrix-type microfluidic device.

产生衔接子修饰的靶核酸分子Generating adaptor-modified target nucleic acid molecules

在某些实施方案中，本发明涉及向包含粘末端的多个靶核酸的每个末端添加衔接子分子的方法。这些实施方案可用于，例如，为了高通量DNA测序的片段产生。可选择衔接子以帮助使用所选的DNA测序平台测序。In certain embodiments, the present invention relates to methods for adding adapter molecules to each end of a plurality of target nucleic acids comprising sticky ends. These embodiments can be used, for example, to generate fragments for high-throughput DNA sequencing. The adapters can be selected to facilitate sequencing using a selected DNA sequencing platform.

在具体实施方案中，这样的方法包括退火衔接子分子到双链靶核酸分子的粘末端以产生退火的衔接子-靶核酸分子。包含粘末端的靶核酸分子可通过任何方便的方法产生。在某些实施方案中，将DNA分子片段化，例如，通过酶促消化、喷雾法、声处理(sonication)等等的任一种。例如，DNA分子可通过用DNA酶诸如DNA酶I消化来片段化，通过热处理结束。不产生粘末端的片段化随后可以是用酶消化片段化的DNA分子以产生粘末端。在具体实施方案中，双链靶核酸分子的粘末端是3'延伸。在消化采用的条件下不具有聚合酶活性的链特异性核酸内切酶可用来产生粘末端。在示例性实施方案中，通过在dNTP不存在下用核酸外切酶III消化5'末端来产生粘末端。In a specific embodiment, such a method includes annealing an adapter molecule to the sticky end of a double-stranded target nucleic acid molecule to produce an annealed adapter-target nucleic acid molecule. The target nucleic acid molecule comprising a sticky end can be produced by any convenient method. In certain embodiments, the DNA molecule is fragmented, for example, by any of enzymatic digestion, spraying, sonication, etc. For example, the DNA molecule can be fragmented by digesting with a DNA enzyme such as DNA enzyme I, and then terminated by heat treatment. The fragmentation that does not produce a sticky end can then be digested with an enzyme to produce a sticky end. In a specific embodiment, the sticky end of the double-stranded target nucleic acid molecule is a 3' extension. A chain-specific endonuclease that does not have polymerase activity under the conditions used for digestion can be used to produce a sticky end. In an exemplary embodiment, a sticky end is produced by digesting the 5' end with an exonuclease III in the absence of dNTPs.

在第一实施方案中，衔接子分子是发夹结构，各自包含：衔接子核苷酸序列，其连接于核苷酸接头，其连接于能够退火到衔接子核苷酸序列并连接于简并尾序列的核苷酸序列。参见图1A。这一实施方案采用两个类型的衔接子分子，其中每个类型包含不同于另一类型的衔接子核苷酸序列(即，第一衔接子核苷酸序列和第二衔接子核苷酸序列)。In a first embodiment, the adapter molecules are hairpin structures, each comprising: an adapter nucleotide sequence linked to a nucleotide linker linked to a nucleotide sequence capable of annealing to the adapter nucleotide sequence and linked to a degenerate tail sequence. See Figure 1A. This embodiment employs two types of adapter molecules, wherein each type comprises an adapter nucleotide sequence that is different from the other type (i.e., a first adapter nucleotide sequence and a second adapter nucleotide sequence).

在第二实施方案中，衔接子分子是双链或单链分子，在每条链上各自包含：第一衔接子核苷酸序列，其连接于核苷酸接头，其连接于第二衔接子核苷酸序列；和简并尾序列，其中双链分子各自包含两个简并尾序列作为粘末端。参见图2A。In a second embodiment, the adapter molecule is a double-stranded or single-stranded molecule, each comprising on each strand: a first adapter nucleotide sequence connected to a nucleotide linker connected to a second adapter nucleotide sequence; and a degenerate tail sequence, wherein each double-stranded molecule comprises two degenerate tail sequences as sticky ends. See Figure 2A.

在某些实施方案中，例如，其中靶核酸分子为高通量DNA测序准备的实施方案中，第一和第二衔接子序列可包含能够被DNA测序引物即测序仪特异性标签1和测序仪特异性标签2特异性结合的引物结合位点。参见图1A和2A。In certain embodiments, for example, embodiments in which the target nucleic acid molecule is prepared for high-throughput DNA sequencing, the first and second adapter sequences can comprise primer binding sites capable of being specifically bound by DNA sequencing primers, namely, sequencer-specific tag 1 and sequencer-specific tag 2. See Figures 1A and 2A.

在所有情形中，简并尾序列可以在衔接子分子的3'末端。衔接子分子的简并尾序列与靶核酸分子上的粘末端的至少一部分基本上互补；即，在采用的条件下衔接子分子能够退火到靶核酸分子。简并尾序列的长度将通常足以帮助这一退火，例如，约10至约20个核苷酸。在某些实施方案中，简并尾序列在其3'末端被保护，例如，用磷酸硫代硫酸酯(phosphothionate)或dUTP保护以保护不受核酸外切酶消化。In all cases, the degenerate tail sequence can be at the 3' end of the adapter molecule. The degenerate tail sequence of the adapter molecule is substantially complementary to at least a portion of the sticky end on the target nucleic acid molecule; that is, the adapter molecule is capable of annealing to the target nucleic acid molecule under the conditions employed. The length of the degenerate tail sequence will generally be sufficient to facilitate this annealing, e.g., from about 10 to about 20 nucleotides. In certain embodiments, the degenerate tail sequence is protected at its 3' end, e.g., with phosphothioate or dUTP to protect from exonuclease digestion.

任选地，衔接子分子可包含一个或多个另外的核苷酸序列。在某些实施方案中，衔接子分子的核苷酸接头部分可包含核酸内切酶位点、条形码核苷酸序列、亲和性标签、和其任何组合。例如，核苷酸接头可包含限制性酶位点、和任选地，至少一个条形码核苷酸序列。Optionally, the adapter molecule may comprise one or more additional nucleotide sequences. In certain embodiments, the nucleotide linker portion of the adapter molecule may comprise an endonuclease site, a barcode nucleotide sequence, an affinity tag, and any combination thereof. For example, the nucleotide linker may comprise a restriction enzyme site and, optionally, at least one barcode nucleotide sequence.

在第一和第二实施方案中，在退火到靶核酸分子后，方法包括填充退火的衔接子-靶核酸分子中的任何缺口(例如利用DNA聚合酶)，和连接退火的衔接子-靶核酸分子中任何邻近的核苷酸序列以产生衔接子修饰的靶核酸分子。在一些实施方案中，粘末端产生和连接可在相同的反应混合物中进行。例如核酸外切酶可在单个反应混合物中与连接酶(例如，热稳定连接酶)和聚合酶(例如，)一起使用。In the first and second embodiments, after annealing to the target nucleic acid molecule, the method includes filling any gaps in the annealed adapter-target nucleic acid molecule (e.g., using a DNA polymerase), and ligating any adjacent nucleotide sequences in the annealed adapter-target nucleic acid molecule to produce an adapter-modified target nucleic acid molecule. In some embodiments, sticky end generation and ligation can be performed in the same reaction mixture. For example, an exonuclease can be used in a single reaction mixture with a ligase (e.g., a thermostable ligase) and a polymerase (e.g., ).

当衔接子分子是发夹结构时，衔接子与靶核酸的连接将退火的衔接子-靶核酸分子转变为单链环状DNA分子，其可形成双链结构，如图1D中所示的。当衔接子分子是单链或双链分子时，衔接子与靶核酸的连接将退火的衔接子-靶核酸分子转变为双链环状DNA分子。当核苷酸接头包含核酸内切酶位点时，方法可另外包括消化单链或双链环状DNA分子以产生线性DNA分子。参见图1D和2D。具体地，可用在核苷酸接头中的位点切割的限制性酶消化双链环状DNA分子以产生线性DNA分子。在具体实施方案中，线性DNA分子包含5'-核苷酸接头的第一部分-第二衔接子核苷酸序列-第一简并尾序列-靶核酸分子-第二简并尾序列-第一衔接子核苷酸序列-核苷酸接头的第二部分-3'。When the adapter molecule is a hairpin structure, the connection of the adapter to the target nucleic acid converts the annealed adapter-target nucleic acid molecule into a single-stranded circular DNA molecule, which can form a double-stranded structure, as shown in Figure 1D. When the adapter molecule is a single-stranded or double-stranded molecule, the connection of the adapter to the target nucleic acid converts the annealed adapter-target nucleic acid molecule into a double-stranded circular DNA molecule. When the nucleotide linker comprises an endonuclease site, the method can additionally include digesting the single-stranded or double-stranded circular DNA molecule to produce a linear DNA molecule. See Figures 1D and 2D. Specifically, the restriction enzyme that can be used to cut the site in the nucleotide linker digests the double-stranded circular DNA molecule to produce a linear DNA molecule. In a specific embodiment, the linear DNA molecule comprises the first part of the 5'-nucleotide linker-the second adapter nucleotide sequence-the first degenerate tail sequence-the target nucleic acid molecule-the second degenerate tail sequence-the first adapter nucleotide sequence-the second part of the nucleotide linker-3'.

在示例性实施方案中，上述方法可通过以下进行：In an exemplary embodiment, the above method can be performed by:

通过以下产生包含粘末端的多个靶核酸分子：A plurality of target nucleic acid molecules comprising sticky ends are generated by:

用DNA酶I消化DNA分子以产生片段化的DNA分子，然后热灭活DNA酶I；digesting the DNA molecules with DNase I to produce fragmented DNA molecules, and then heat-inactivating the DNase I;

在脱氧核苷酸不存在下用具有5'至3'核酸外切酶活性的核酸酶(诸如核酸外切酶III)消化片段化的DNA分子以产生多个具有粘末端的靶核酸分子；digesting the fragmented DNA molecules with a nuclease having 5' to 3' exonuclease activity (such as exonuclease III) in the absence of deoxynucleotides to generate a plurality of target nucleic acid molecules having sticky ends;

退火所述衔接子到多个靶核酸分子的粘末端，其中衔接子的核苷酸接头包含核酸内切酶位点；annealing the adaptor to the sticky ends of the plurality of target nucleic acid molecules, wherein the nucleotide linker of the adaptor comprises an endonuclease site;

在包含聚合酶和连接酶的单个反应中填充退火的衔接子-靶核酸分子中的任何缺口并连接任何邻近的核苷酸序列以产生环状DNA分子；和filling any gaps in the annealed adapter-target nucleic acid molecules and ligating any adjacent nucleotide sequences to produce a circular DNA molecule in a single reaction comprising a polymerase and a ligase; and

用在核酸内切酶位点切割的核酸内切酶消化环状DNA分子以产生线性DNA分子。The circular DNA molecule is digested with an endonuclease that cuts at the endonuclease site to produce a linear DNA molecule.

在具体实施方案中，加入衔接子分子到多个靶核酸的每个末端的方法可包括通过任何可得的方法测序衔接子修饰的靶核酸分子，诸如任何可得的高通量DNA测序技术。In particular embodiments, the method of adding an adaptor molecule to each end of a plurality of target nucleic acids can include sequencing the adaptor-modified target nucleic acid molecules by any available method, such as any available high-throughput DNA sequencing technology.

掺入核酸序列到靶核酸中Incorporation of nucleic acid sequences into target nucleic acids

掺入一个或多个核苷酸序列到靶核酸中的反应可利用除了退火到靶核酸的部分以外还包含一个或多个核酸序列的两个或多个引物进行。这些部分的一个或多个可包含随机序列以掺入核酸序列到样品中的基本上所有核酸中。可选地或此外，这些部分的一个或多个可以是对存在的多个或所有核酸共有的一个或多个序列特异性的。在其他实施方案中，引物包含对一个或多个特定靶核酸特异性的部分。核酸序列可利用少至两个引物被掺入。然而，多个实施方案采用三个、四个、五个、或六个或更多个引物，如在以下更详细描述的。以下关于核酸扩增讨论此类反应；然而，本领域技术人员将容易领会，以下讨论的策略可在其他类型的反应例如聚合酶延伸和连接中采用。The reaction of incorporating one or more nucleotide sequences into the target nucleic acid can be carried out using two or more primers that also include one or more nucleic acid sequences in addition to the portion that anneals to the target nucleic acid. One or more of these portions can include random sequences to incorporate the nucleic acid sequence into substantially all nucleic acids in the sample. Alternatively or in addition, one or more of these portions can be specific to one or more sequences common to multiple or all nucleic acids present. In other embodiments, the primer includes a portion specific to one or more specific target nucleic acids. The nucleic acid sequence can be incorporated using as few as two primers. However, multiple embodiments employ three, four, five, or six or more primers, as described in more detail below. This type of reaction is discussed below with respect to nucleic acid amplification; however, those skilled in the art will readily appreciate that the strategies discussed below can be employed in other types of reactions, such as polymerase extension and ligation.

三引物方法Three-primer method

在具体实施方案中，本发明提供了用于将多个(例如至少三个)选择的核苷酸序列掺入到一个或多个靶核酸中的扩增方法。在一些实施方案中，这种方法包括扩增在多个样品中的多个靶核酸。在示意性实施方案中，可以在两个或多个不同样品的每一个中扩增同一组靶核酸。这些样品可以在任何方面彼此不同，例如这些样品可以是来自不同组织、受试者、环境来源等。可以使用至少三个引物来扩增每个靶核酸，即：正向和反向扩增引物，每个引物包括靶特异性部分，并且一个或两个引物包括核苷酸标签(例如，第一和第二核苷酸标签)。这些靶特异性部分可以在适当的退火条件下特异性地退火到靶上。用于正向引物的核苷酸标签可以具有与用于反向引物的核苷酸标签相同或不同的序列。总体上，这些核苷酸标签在这些靶特异性部分的5'。第三个引物是条形码引物，该条形码引物包括条形码核苷酸序列以及第一和/或第二核苷酸标签特异性部分。条形码核苷酸序列是选择用于编码关于当条形码引物被用于扩增反应时所生成的扩增子的信息的序列。标签特异性部分可以特异性地退火到正向和反向引物中的一个或两个核苷酸标签上。总体上条形码引物在靶特异性部分的5'。In a specific embodiment, the present invention provides an amplification method for incorporating multiple (e.g., at least three) selected nucleotide sequences into one or more target nucleic acids. In some embodiments, this method includes amplifying multiple target nucleic acids in multiple samples. In an illustrative embodiment, the same set of target nucleic acids can be amplified in each of two or more different samples. These samples can differ from each other in any aspect, for example, these samples can be from different tissues, subjects, environmental sources, etc. At least three primers can be used to amplify each target nucleic acid, namely: forward and reverse amplification primers, each primer including a target-specific portion, and one or both primers including a nucleotide tag (e.g., a first and a second nucleotide tag). These target-specific portions can be specifically annealed to the target under appropriate annealing conditions. The nucleotide tag used for the forward primer can have a sequence that is the same or different from the nucleotide tag used for the reverse primer. Generally, these nucleotide tags are 5' to these target-specific portions. The third primer is a barcode primer that includes a barcode nucleotide sequence and a first and/or second nucleotide tag-specific portion. The barcode nucleotide sequence is a sequence selected to encode information about the amplicon generated when the barcode primer is used in an amplification reaction. The tag-specific portion can specifically anneal to one or both nucleotide tags in the forward and reverse primers. Generally the barcode primer is 5' to the target-specific portion.

条形码引物典型地以超过一个或多个正向和/或反向或(内侧)引物的量存在于扩增混合物中。更确切地说，如果条形码引物退火到正向引物中的核苷酸标签上，总体上该条形码引物以超过正向引物的量存在。如果条形码引物退火到反向引物中的核苷酸标签上，总体上该条形码引物以超过反向引物的量存在。在示意性实施方案中，在每种情况中，在扩增混合物中的第三引物即反向引物或正向引物对应地能够以大致类似于条形码引物的浓度存在。总体上，条形码引物以实质性地过量形式存在。例如，扩增混合物中条形码引物的浓度可以是相对于该一个或多个正向和/或反向引物的浓度的至少2倍、至少4倍、至少5倍、至少10倍、至少15倍、至少20倍、至少25倍、至少30倍、至少35倍、至少40倍、至少45倍、至少50倍、至少100倍、至少500倍、至少10³倍、至少5X10³倍、至少10⁴倍、至少5X10⁴倍、至少10⁵倍、至少5X10⁵倍、至少10⁶倍或更高。此外，条形码引物的浓度超过量可以落在以上述数值的任何一项作为端点的任何范围内(例如，2倍至10⁵倍)。在示意性实施方案中，当条形码引物具有对于正向引物上的核苷酸标签具有特异性的标签特异性部分时，该正向引物能够以皮摩尔至纳摩尔的浓度存在，例如约5pM至500nM、约5pM至100nM、约5pM至50nM、约5pM至10nM、约5pM至5nM、约10pM至1nM、约50pM至约500pM、约100pM或以这些数值任何一项作为端点的任何其他范围(例如，10pM至50pM)。适当地，可与正向引物的这些浓度中任何一项相组合使用的条形码引物的示例性浓度包括约10nM至约10μM、约25nM至约7.5μM、约50nM至约5μM、约75nM至约2.5μM、约100nM至约1μM、约250nM至约750nM、约500nM或以这些数值中任何一项作为端点的任何其他范围(例如，100nM至500nM)。在使用这类浓度的正向和条形码引物的扩增反应中，反向引物具有与条形码引物处于同一数量级的浓度(例如，在约10倍之内、在约5倍之内、或相等)。The barcode primer is typically present in the amplification mixture in an amount that exceeds one or more forward and/or reverse or (inner) primers. More specifically, if the barcode primer anneals to the nucleotide tag in the forward primer, the barcode primer is generally present in an amount that exceeds the forward primer. If the barcode primer anneals to the nucleotide tag in the reverse primer, the barcode primer is generally present in an amount that exceeds the reverse primer. In an illustrative embodiment, in each case, the third primer in the amplification mixture, i.e., the reverse primer or the forward primer, can be present in a concentration roughly similar to that of the barcode primer, respectively. Generally, the barcode primer is present in substantial excess. For example, the concentration of the barcode primer in the amplification mixture can be at least 2-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 10 3-fold, at least 5×10^3- fold, at least^{10 4-} fold, at least 5×10 4-fold, at least 10⁵ -fold, at least 5^× 10^5- fold, at least 10⁶ -fold, or more relative to the concentration of the one or more forward and/or reverse primers. In addition, the excess concentration of the barcode primer can fall within any range (e.g., 2-fold to 10^5- fold) with any of the above values as endpoints. In illustrative embodiments, when the barcode primer has a tag-specific portion that is specific for a nucleotide tag on the forward primer, the forward primer can be present at a concentration in the range of picomolar to nanomolar, e.g., about 5 pM to 500 nM, about 5 pM to 100 nM, about 5 pM to 50 nM, about 5 pM to 10 nM, about 5 pM to 5 nM, about 10 pM to 1 nM, about 50 pM to about 500 pM, about 100 pM, or any other range having any of these values as endpoints (e.g., 10 pM to 50 pM). Suitably, exemplary concentrations of the barcode primer that can be used in combination with any of these concentrations of the forward primer include about 10 nM to about 10 μM, about 25 nM to about 7.5 μM, about 50 nM to about 5 μM, about 75 nM to about 2.5 μM, about 100 nM to about 1 μM, about 250 nM to about 750 nM, about 500 nM, or any other range with any of these values as endpoints (e.g., 100 nM to 500 nM). In an amplification reaction using such concentrations of the forward and barcode primers, the reverse primer has a concentration that is on the same order of magnitude as the barcode primer (e.g., within about 10-fold, within about 5-fold, or equal).

可以使每个扩增混合物经受扩增作用从而生成包括加标签的靶核苷酸序列的靶扩增子，各自包括位于该靶核苷酸序列侧翼的第一和第二核苷酸标签，以及位于该靶扩增子的5’或3’末端上的至少一个条形码核苷酸序列(相对于靶扩增子的一个链)。在某些实施方案中，选择第一和第二核苷酸标签和/或条形码核苷酸序列以便避免实质性地退火到靶核酸上。在这类实施方案中，加标签的靶核苷酸序列可以包括具有下面元件的分子:5’-(条形码核苷酸序列)-(来自正向引物的第一核苷酸标签)-(靶核苷酸序列)-(来自反向引物的第二核苷酸标签序列)-3’或5’-(来自正向引物的第一核苷酸标签)-(靶核苷酸序列)-(来自反向引物的第二核苷酸标签序列)-(条形码核苷酸序列)-3'。Each amplification mixture can be subjected to amplification to generate target amplicons comprising a tagged target nucleotide sequence, each comprising a first and a second nucleotide tag flanking the target nucleotide sequence, and at least one barcode nucleotide sequence located at the 5' or 3' end of the target amplicon (relative to one strand of the target amplicon). In certain embodiments, the first and second nucleotide tags and/or the barcode nucleotide sequence are selected so as to avoid substantial annealing to the target nucleic acid. In such embodiments, the tagged target nucleotide sequence can comprise a molecule having the following elements: 5'-(barcode nucleotide sequence)-(first nucleotide tag from the forward primer)-(target nucleotide sequence)-(second nucleotide tag sequence from the reverse primer)-3' or 5'-(first nucleotide tag from the forward primer)-(target nucleotide sequence)-(second nucleotide tag sequence from the reverse primer)-(barcode nucleotide sequence)-3'.

四引物方法Four-primer method

在一些实施方案中，可以使用多于三个引物以将希望的元件加入靶核苷酸序列中。例如，可以使用四个引物来生成具有如上讨论的相同的元件加上任选的另外的条形码的分子，例如5’-(条形码核苷酸序列)-(来自正向引物的第一核苷酸标签)-(靶核苷酸序列)-(来自反向引物的第二核苷酸标签)-(另外的条形码核苷酸序列)-3'。在示例性四引物实施方案中，正向引物包括靶特异性部分以及第一核苷酸标签，并且反向引物包括靶特异性部分以及第二核苷酸标签。总之，这两个引物构成“内侧引物”。剩下的两个引物是“外侧引物”，其退火到存在于内侧引物中的第一和第二核苷酸标签上。一个外侧引物是条形码引物，如以上所述的。第二外侧引物可以包括第二标签特异性部分和另外的条形码核苷酸序列，即，其可以是第二条形码引物。In some embodiments, more than three primers can be used to add desired elements to the target nucleotide sequence. For example, four primers can be used to generate molecules with the same elements as discussed above plus an optional additional barcode, such as 5'-(barcode nucleotide sequence)-(first nucleotide tag from the forward primer)-(target nucleotide sequence)-(second nucleotide tag from the reverse primer)-(additional barcode nucleotide sequence)-3'. In an exemplary four-primer embodiment, the forward primer includes a target-specific portion and a first nucleotide tag, and the reverse primer includes a target-specific portion and a second nucleotide tag. In short, these two primers constitute "inside primers." The remaining two primers are "outside primers," which anneal to the first and second nucleotide tags present in the inside primer. One outside primer is a barcode primer, as described above. The second outside primer can include a second tag-specific portion and an additional barcode nucleotide sequence, that is, it can be a second barcode primer.

在一个或多个扩增反应中可以进行扩增以掺入来自超过三个引物的元件。例如，可以在其中存在所有四个引物的一个扩增反应中进行四引物扩增。作为替代方案，可以例如在两个扩增反应中进行四引物扩增：一个用来掺入内侧引物并且一个不同的扩增反应用于掺入外侧引物。当所有四个引物都存在于一个扩增反应中时，外侧引物总体上是以过量形式存在于反应混合物中。在一步、四引物扩增反应中，对于条形码引物相对于正向和/或反向引物而言如上给出的相对浓度值还应用到外侧引物相对于内侧引物的浓度上。In one or more amplified reactions, amplification can be carried out to mix the element from more than three primers. For example, a four-primer amplification can be carried out in an amplified reaction in which there are all four primers. As an alternative, a four-primer amplification can be carried out in two amplified reactions: one is used to mix an inner primer and a different amplified reaction is used to mix an outer primer. When all four primers were present in an amplified reaction, the outer primer was generally present in the reaction mixture in excessive form. In one step, four-primer amplified reactions, the relative concentration value given as above for barcode primers with respect to forward and/or reverse primer also applies to the outer primer with respect to the concentration of the inner primer.

组合方法Combination Methods

在四引物扩增反应的示例性实施方案中，外侧引物的每一个包含独特的条形码。例如，一个条形码引物可以由以下元件构成：5’-(第一条形码核苷酸序列)-(第一核苷酸标签)-3'，并且该第二条形码引物可以由以下元件构成：5’-(第二条形码核苷酸序列)-(第二核苷酸标签)-3'。在这个实施方案中，可以使一定数量(J)的第一条形码引物与一定数量(K)的第二条形码引物组合从而生成JxK个独特的扩增产物。In an exemplary embodiment of a four-primer amplification reaction, each outer primer contains a unique barcode. For example, one barcode primer can be composed of the following elements: 5'-(first barcode nucleotide sequence)-(first nucleotide tag)-3', and the second barcode primer can be composed of the following elements: 5'-(second barcode nucleotide sequence)-(second nucleotide tag)-3'. In this embodiment, a certain number (J) of first barcode primers can be combined with a certain number (K) of second barcode primers to generate JxK unique amplification products.

在本发明的进一步示例性实施方案中，可以将多于4个引物结合到一个单一反应中从而附加条形码核苷酸序列和核苷酸标签的不同组合。例如，如上所述可以使包含以下元件的外侧条形码引物：5’-(第一条形码核苷酸序列)-(第一核苷酸标签)-3'、5-(第一条形码核苷酸序列)-(第二核苷酸标签)-3'、5’-(第二条形码核苷酸序列)-(第一核苷酸标签)-3'、5’-(第二条形码核苷酸序列)-(第二核苷酸标签)-3'，与内侧靶特异性引物组合从而生成包含条形码引物与所希望的扩增子序列的所有组合的扩增产物池。In a further exemplary embodiment of the present invention, more than four primers can be combined into a single reaction to attach different combinations of barcode nucleotide sequences and nucleotide tags. For example, as described above, outer barcode primers comprising the following elements: 5'-(first barcode nucleotide sequence)-(first nucleotide tag)-3', 5-(first barcode nucleotide sequence)-(second nucleotide tag)-3', 5'-(second barcode nucleotide sequence)-(first nucleotide tag)-3', 5'-(second barcode nucleotide sequence)-(second nucleotide tag)-3' can be combined with inner target-specific primers to generate a pool of amplification products comprising all combinations of barcode primers and desired amplicon sequences.

在本发明的其他示例性实施方案中，可以使上述组合或对于本领域普通技术人员而言是明显的其他组合中的任何一项中的外侧条形码引物与多于一对的具有相同第一和第二核苷酸标签序列的靶引物序列组合。例如，如上所述，可以使包含与同一第一核苷酸标签组合的高达十个不同靶特异性正向引物序列以及与同一第二核苷酸标签组合的高达十个不同靶特异性反向引物序列的内侧引物与高达2个或高达4个外侧条形码引物组合从而生成多个扩增产物。在不同实施方案中，可以使携带相同第一核苷酸标签以及第二核苷酸标签的至少10个、至少20个、至少50个、至少100个、至少200个、至少500个、至少1000个、至少2000个、至少5000个或至少10000个不同靶特异性引物对与高达2个或高达4个外侧条形码引物组合从而生成多个扩增产物。In other exemplary embodiments of the present invention, outer barcode primers from any of the above combinations, or other combinations apparent to one of ordinary skill in the art, can be combined with more than one pair of target primer sequences having the same first and second nucleotide tag sequences. For example, as described above, inner primers comprising up to ten different target-specific forward primer sequences combined with the same first nucleotide tag and up to ten different target-specific reverse primer sequences combined with the same second nucleotide tag can be combined with up to two or up to four outer barcode primers to generate multiple amplification products. In various embodiments, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 5000, or at least 10,000 different target-specific primer pairs carrying the same first and second nucleotide tags can be combined with up to two or up to four outer barcode primers to generate multiple amplification products.

双向组合方法Two-way combination method

在四引物扩增反应的示例性的实施方案中，内侧和外侧引物可各自包含独特的条形码，从而扩增产生在所得的扩增子的每个末端的条形码组合。当扩增子将被测序时这一方法是有用的，因为条形码组合可从序列的任一末端读取。例如，可采用四引物以产生具有以下元件的分子：5'-第二条形码核苷酸序列-第一核苷酸标签序列-第一条形码核苷酸序列-靶核苷酸序列-第一条形码核苷酸序列-第二核苷酸标签序列-第二条形码核苷酸序列-3'。在示例性的四引物实施方案中，两个内侧引物可包含：In an exemplary embodiment of a four-primer amplification reaction, the inner and outer primers can each contain a unique barcode, thereby amplifying a barcode combination at each end of the resulting amplicon. This approach is useful when the amplicon is to be sequenced, because the barcode combination can be read from either end of the sequence. For example, a four-primer amplification reaction can be used to generate a molecule having the following elements: 5'-second barcode nucleotide sequence-first nucleotide tag sequence-first barcode nucleotide sequence-target nucleotide sequence-first barcode nucleotide sequence-second nucleotide tag sequence-second barcode nucleotide sequence-3'. In an exemplary four-primer embodiment, the two inner primers can contain:

正向、内侧引物，包含第一核苷酸标签、第一条形码核苷酸序列和靶特异性部分；和a forward, inner primer comprising a first nucleotide tag, a first barcode nucleotide sequence, and a target-specific portion; and

反向、内侧引物，包含靶特异性部分、第一条形码核苷酸序列和第二核苷酸标签。两个外侧引物可包含：The reverse, inner primer comprises a target-specific portion, a first barcode nucleotide sequence, and a second nucleotide tag. The two outer primers may comprise:

正向、外侧引物，包含第二条形码核苷酸序列和第一核苷酸标签特异性部分；和a forward, outer primer comprising a second barcode nucleotide sequence and a first nucleotide tag-specific portion; and

反向、外侧引物，包含第二核苷酸标签特异性部分和第二条形码核苷酸序列。如以上讨论的，如果内侧和外侧引物被包含在相同的反应混合物中，外侧引物优选地以过量存在。The reverse, outer primer comprises a second nucleotide tag-specific portion and a second barcode nucleotide sequence. As discussed above, if the inner and outer primers are included in the same reaction mixture, the outer primer is preferably present in excess.

除了内侧和外侧引物以外还采用"填充"引物的六引物扩增方法中可产生元件的类似组合。如此，例如，两个内侧引物可包含：Similar combinations of elements can be generated in a six-primer amplification method that employs "filler" primers in addition to the inner and outer primers. Thus, for example, the two inner primers may comprise:

正向、内侧引物，包含第一核苷酸标签和靶特异性部分；和a forward, inner primer comprising a first nucleotide tag and a target-specific portion; and

反向、内侧引物，包含靶特异性部分和第二核苷酸标签。两个填充引物可包含：A reverse, inner primer containing a target-specific portion and a second nucleotide tag. The two filler primers may contain:

正向、填充引物，包含第三核苷酸标签、第一条形码核苷酸序列和第一核苷酸标签特异性部分；和a forward, filler primer comprising a third nucleotide tag, a first barcode nucleotide sequence, and a portion specific for the first nucleotide tag; and

反向、填充引物，包含第二核苷酸标签特异性部分、第一条形码核苷酸序列、第四核苷酸标签。两个外侧引物可包含：The reverse, filler primer contains the second nucleotide tag-specific portion, the first barcode nucleotide sequence, and the fourth nucleotide tag. The two outer primers may contain:

正向、外侧引物，包含第二条形码核苷酸序列和第三核苷酸标签特异性部分；和a forward, outer primer comprising a second barcode nucleotide sequence and a third nucleotide tag-specific portion; and

反向、外侧引物，包含第四核苷酸标签特异性部分和第二条形码核苷酸序列。核酸扩增产生包含以下元件的扩增子：5'-第二条形码核苷酸序列-第三核苷酸标签序列-第一条形码核苷酸序列-第一核苷酸标签序列-靶核苷酸序列-第二核苷酸标签序列-第一条形码核苷酸序列-第四核苷酸标签序列-第二条形码核苷酸序列-3'。扩增可在一个、两个、三个扩增反应中进行。例如，所有三个引物对可被包含在一个反应中。可选地，可进行两个反应，例如，第一反应包括内侧和填充引物，第二反应仅包括外侧引物；或第一反应仅包括内侧引物，随后第二反应包括填充和外侧引物。当存在多于一个引物对时，相对于其他对为"外侧"对的引物对优选地以过量存在，如以上讨论的。如此，如果内侧和填充引物被包含在反应混合物中，填充引物优选地以过量存在，如果填充和外侧引物被包含在反应混合物中，外侧引物优选地以过量存在。当所有三个引物对被包含在单个反应中时，填充引物可以内侧引物和外侧引物的浓度之间的中间浓度存在。A reverse, outer primer comprises a portion specific for the fourth nucleotide tag and a second barcode nucleotide sequence. Nucleic acid amplification produces an amplicon comprising the following elements: 5'-second barcode nucleotide sequence-third nucleotide tag sequence-first barcode nucleotide sequence-first nucleotide tag sequence-target nucleotide sequence-second nucleotide tag sequence-first barcode nucleotide sequence-fourth nucleotide tag sequence-second barcode nucleotide sequence-3'. Amplification can be performed in one, two, or three amplification reactions. For example, all three primer pairs can be included in a single reaction. Alternatively, two reactions can be performed, e.g., a first reaction including inner and filler primers and a second reaction including only outer primers; or a first reaction including only inner primers, followed by a second reaction including filler and outer primers. When more than one primer pair is present, the primer pair that is the "outer" pair relative to the other pairs is preferably present in excess, as discussed above. Thus, if inner and filler primers are included in the reaction mixture, the filler primer is preferably present in excess, and if filler and outer primers are included in the reaction mixture, the outer primer is preferably present in excess. When all three primer pairs are included in a single reaction, the stuffer primer may be present at a concentration intermediate between the concentrations of the inner and outer primers.

在上述四引物和六引物扩增方法的某些实施方案中，例如，当反应中产生的分子将被进行DNA测序时，外侧引物可另外包含能够被DNA测序引物结合的第一和第二引物结合位点。例如，四引物反应可产生包含以下的加标签的靶核苷酸序列：5'-第一引物结合位点-第二条形码核苷酸序列-第一核苷酸标签序列-第一条形码核苷酸序列-靶核苷酸序列-第一条形码核苷酸序列-第二核苷酸标签序列-第二条形码核苷酸序列-第二引物结合位点-3'。这一实施方案提供的益处是，条形码组合可从分子任一末端的测序读取中确定。类似地，六引物反应可产生包含以下的加标签的靶核苷酸序列：5'-第一引物结合位点-第二条形码核苷酸序列-第三核苷酸标签序列-第一条形码核苷酸序列-第一核苷酸标签序列-靶核苷酸序列-第二核苷酸标签序列-第一条形码核苷酸序列-第四核苷酸标签序列-第二条形码核苷酸序列-第二引物结合位点-3'。In certain embodiments of the above-described four-primer and six-primer amplification methods, for example, when the molecules produced in the reaction are to be subjected to DNA sequencing, the outer primers may additionally include first and second primer binding sites capable of being bound by DNA sequencing primers. For example, a four-primer reaction can produce a tagged target nucleotide sequence comprising the following: 5′-first primer binding site-second barcode nucleotide sequence-first nucleotide tag sequence-first barcode nucleotide sequence-target nucleotide sequence-first barcode nucleotide sequence-second nucleotide tag sequence-second barcode nucleotide sequence-second primer binding site-3′. This embodiment provides the advantage that the barcode combination can be determined from sequencing reads at either end of the molecule. Similarly, a six-primer reaction can produce a tagged target nucleotide sequence comprising the following: 5′-first primer binding site-second barcode nucleotide sequence-third nucleotide tag sequence-first barcode nucleotide sequence-first nucleotide tag sequence-target nucleotide sequence-second nucleotide tag sequence-first barcode nucleotide sequence-fourth nucleotide tag sequence-second barcode nucleotide sequence-second primer binding site-3′.

基于组合连接的加标签Labeling based on combined connections

在某些实施方案中，本发明包括用于将多个靶核苷酸序列组合加标签(例如，条形码化)的基于连接的方法。该方法采用来源于靶核酸的多个加标签的靶核苷酸序列。每个加标签的靶核苷酸序列包含核酸内切酶位点和第一条形码核苷酸序列。多个加标签的靶核苷酸序列包含相同的核酸内切酶位点、但N个不同的第一条形码核苷酸序列，其中N是大于1的整数。In certain embodiments, the present invention includes a ligation-based method for combinatorially tagging (e.g., barcoding) a plurality of target nucleotide sequences. The method employs a plurality of tagged target nucleotide sequences derived from a target nucleic acid. Each tagged target nucleotide sequence comprises an endonuclease site and a first barcode nucleotide sequence. The plurality of tagged target nucleotide sequences comprises the same endonuclease site but N different first barcode nucleotide sequences, where N is an integer greater than 1.

用对核酸内切酶位点特异性的核酸内切酶切割加标签的靶核苷酸序列以产生多个具有粘末端的、加标签的靶核苷酸序列。然后在第一反应混合物中连接多个衔接子到加标签的靶核苷酸序列。多个衔接子包含第二条形码核苷酸序列和与多个具有粘末端的、加标签的靶核苷酸序列互补的粘末端。而且，多个衔接子包含M个不同的第二条形码核苷酸序列，其中M是大于1的整数。连接产生多个组合加标签的靶核苷酸序列，各自包含第一和第二条形码核苷酸序列，其中多个包含NxM个不同的第一和第二条形码组合。The tagged target nucleotide sequence is cleaved with an endonuclease specific for the endonuclease site to produce a plurality of tagged target nucleotide sequences having sticky ends. A plurality of adapters are then ligated to the tagged target nucleotide sequences in a first reaction mixture. The plurality of adapters comprises a second barcode nucleotide sequence and sticky ends complementary to the plurality of tagged target nucleotide sequences having sticky ends. Furthermore, the plurality of adapters comprises M different second barcode nucleotide sequences, where M is an integer greater than 1. Ligation produces a plurality of combinatorial tagged target nucleotide sequences, each comprising a first and a second barcode nucleotide sequence, wherein a plurality comprises N x M different first and second barcode combinations.

在某些实施方案中，核酸内切酶位点邻近加标签的靶核苷酸序列中的第一条形码核苷酸序列。在此类实施方案的变化形式中，第二条形码核苷酸序列邻近衔接子中的互补粘末端。在具体实施方案中，例如，组合加标签的靶核苷酸序列包含由少于5个核苷酸分隔的第一和第二条形码核苷酸序列。In certain embodiments, the endonuclease site is adjacent to the first barcode nucleotide sequence in the tagged target nucleotide sequence. In variations of such embodiments, the second barcode nucleotide sequence is adjacent to the complementary cohesive end in the adaptor. In specific embodiments, for example, the combinatorially tagged target nucleotide sequence comprises a first and a second barcode nucleotide sequence separated by fewer than 5 nucleotides.

在具体实施方案中，例如，当组合加标签的靶核苷酸序列打算用于测序时，加标签的靶核苷酸序列可包含第一和第二引物结合位点，其可具有以下布置的任一种：5'-核酸内切酶位点-第一条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点；和5'-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一条形码核苷酸序列-核酸内切酶位点-3'。为了帮助测序，第一和第二引物结合位点可以是DNA测序引物的结合位点。在此类实施方案的变化形式中，组合加标签的核苷酸序列可包含以以下布置之一的第二条形码核苷酸序列：5'-第二条形码核苷酸序列-第一条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点；或5'-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一条形码核苷酸序列-第二条形码核苷酸序列-3'。In specific embodiments, for example, when the combinatorially tagged target nucleotide sequence is intended for sequencing, the tagged target nucleotide sequence can include first and second primer binding sites, which can have either of the following arrangements: 5'-endonuclease site-first barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site; and 5'-first primer binding site-target nucleotide sequence-second primer binding site-first barcode nucleotide sequence-endonuclease site-3'. To facilitate sequencing, the first and second primer binding sites can be binding sites for DNA sequencing primers. In variations of such embodiments, the combinatorially tagged nucleotide sequence can include the second barcode nucleotide sequence in one of the following arrangements: 5'-second barcode nucleotide sequence-first barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site; or 5'-first primer binding site-target nucleotide sequence-second primer binding site-first barcode nucleotide sequence-second barcode nucleotide sequence-3'.

可用于这一方法的加标签的靶核苷酸序列可通过任何方便的手段制备，诸如，例如，通过连接衔接子到多个靶核酸，其中衔接子包含：第一衔接子，包含核酸内切酶位点、第一条形码核苷酸序列、第一引物结合位点和粘末端；和第二衔接子，包含第二引物结合位点和粘末端。Tagged target nucleotide sequences useful in this method can be prepared by any convenient means, such as, for example, by ligating adaptors to multiple target nucleic acids, wherein the adaptors comprise: a first adaptor comprising a nuclease site, a first barcode nucleotide sequence, a first primer binding site, and sticky ends; and a second adaptor comprising a second primer binding site and sticky ends.

在一些实施方案中，有利地在加标签的靶核苷酸序列中包含一个或多个另外的核苷酸序列，例如，以帮助操作和/或鉴定。如此，加标签的靶核苷酸序列可包含第一另外的核苷酸序列，具有选自以下的布置：5'-核酸内切酶位点-第一条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一另外的核苷酸序列；和/或5'-第一另外的核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一条形码核苷酸序列-核酸内切酶位点-3'。例如，在Illumina测序中，流动小室结合序列(例如，PE1和PE2)被掺入在待测序的DNA模板的任一末端。在本方法中，加标签的靶核苷酸序列可包含一个流动小室结合序列作为第一另外的核苷酸序列，且另一个流动小室结合序列可经由衔接子被引入。参见，例如，图5A-B。如此，本方法可采用包含第二另外的核苷酸序列并具有以下布置的衔接子：5'-第二另外的核苷酸序列-第二条形码核苷酸序列-互补粘末端-3'。在这种情形中，连接衔接子到包含第一另外的核苷酸序列的上述加标签的靶核苷酸序列产生组合加标签的靶核苷酸序列，包含：5'-第二另外的核苷酸序列-第二条形码核苷酸序列-第一条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一另外的核苷酸序列；和/或5'-第二另外的核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一条形码核苷酸序列-第二条形码核苷酸序列-第一另外的核苷酸序列-3'。在这一实施方案的变化形式中，第一和/或第二另外的核苷酸序列包含引物结合位点。In some embodiments, it is advantageous to include one or more additional nucleotide sequences in the tagged target nucleotide sequence, for example, to aid in manipulation and/or identification. Thus, the tagged target nucleotide sequence may comprise a first additional nucleotide sequence having an arrangement selected from the following: 5'-endonuclease site-first barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first additional nucleotide sequence; and/or 5'-first additional nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first barcode nucleotide sequence-endonuclease site-3'. For example, in Illumina sequencing, flow cell binding sequences (e.g., PE1 and PE2) are incorporated at either end of the DNA template to be sequenced. In the present method, the tagged target nucleotide sequence may comprise one flow cell binding sequence as the first additional nucleotide sequence, and another flow cell binding sequence may be introduced via an adapter. See, for example, Figures 5A-B. Thus, the present method can employ an adapter comprising a second additional nucleotide sequence and having the following arrangement: 5′-second additional nucleotide sequence-second barcode nucleotide sequence-complementary cohesive end-3′. In this case, ligating the adapter to the aforementioned tagged target nucleotide sequence comprising a first additional nucleotide sequence produces a combinatorially tagged target nucleotide sequence comprising: 5′-second additional nucleotide sequence-second barcode nucleotide sequence-first barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first additional nucleotide sequence; and/or 5′-second additional nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first barcode nucleotide sequence-second barcode nucleotide sequence-first additional nucleotide sequence-3′. In variations of this embodiment, the first and/or second additional nucleotide sequence comprises a primer binding site.

包含第一另外的核苷酸序列的加标签的靶核苷酸序列可通过任何方便的手段制备，诸如，例如，通过连接衔接子到多个靶核酸，其中衔接子包含：第一衔接子，包含核酸内切酶位点、第一条形码核苷酸序列、第一引物结合位点和粘末端；和第二衔接子，包含第一另外的核苷酸序列、第二引物结合位点和粘末端。Tagged target nucleotide sequences comprising a first additional nucleotide sequence can be prepared by any convenient means, such as, for example, by ligating adaptors to a plurality of target nucleic acids, wherein the adaptors comprise: a first adaptor comprising a nuclease site, a first barcode nucleotide sequence, a first primer binding site, and sticky ends; and a second adaptor comprising the first additional nucleotide sequence, a second primer binding site, and sticky ends.

基于组合插入诱变的加标签Tagging based on combinatorial insertional mutagenesis

组合加标签还可利用插入诱变进行。在某些实施方案中，多个靶核苷酸序列的组合加标签通过以下进行：退火多个条形码引物到来源于靶核酸的多个加标签的靶核苷酸序列，然后在第一反应混合物中扩增加标签的靶核苷酸序列以产生多个组合加标签的靶核苷酸序列，各自包含第一和第二条形码核苷酸序列，其中多个包含NxM个不同的第一和第二条形码组合。Combinatorial tagging can also be performed using insertional mutagenesis. In certain embodiments, combinatorial tagging of a plurality of target nucleotide sequences is performed by annealing a plurality of barcode primers to a plurality of tagged target nucleotide sequences derived from a target nucleic acid, and then amplifying the tagged target nucleotide sequences in a first reaction mixture to generate a plurality of combinatorially tagged target nucleotide sequences, each comprising a first and a second barcode nucleotide sequence, wherein the plurality comprises NxM different first and second barcode combinations.

在具体实施方案中，每个加标签的靶核苷酸序列包含在一个末端的核苷酸标签、和第一条形码核苷酸序列，其中多个加标签的靶核苷酸序列包含相同的核苷酸标签、但N个不同的第一条形码核苷酸序列，其中N是大于1的整数。在此类实施方案的变化形式中，第一条形码核苷酸序列被靶核苷酸序列与核苷酸标签间隔。每个条形码引物包含：第一标签特异性部分，连接于第二条形码核苷酸序列，其自身连接于第二标签特异性部分，其中多个条形码引物各自包含相同的第一和第二标签特异性部分、但M个不同的第二条形码核苷酸序列，其中M是大于1的整数。条形码引物的第一标签特异性部分退火到核苷酸标签的5'部分，且条形码引物的第二标签特异性部分退火到核苷酸标签的邻近的3'部分(an adjacent 3'portion of the nucleotide tag)，且第二条形码核苷酸序列不退火到核苷酸标签，在退火的第一和第二标签特异性部分之间形成环。In a specific embodiment, each tagged target nucleotide sequence comprises a nucleotide tag at one end and a first barcode nucleotide sequence, wherein a plurality of tagged target nucleotide sequences comprise the same nucleotide tag but N different first barcode nucleotide sequences, where N is an integer greater than 1. In variations of such embodiments, the first barcode nucleotide sequence is separated from the nucleotide tag by the target nucleotide sequence. Each barcode primer comprises: a first tag-specific portion linked to a second barcode nucleotide sequence, which itself is linked to the second tag-specific portion, wherein the plurality of barcode primers each comprise the same first and second tag-specific portions but M different second barcode nucleotide sequences, where M is an integer greater than 1. The first tag-specific portion of the barcode primer anneals to the 5' portion of the nucleotide tag, and the second tag-specific portion of the barcode primer anneals to an adjacent 3' portion of the nucleotide tag, and the second barcode nucleotide sequence does not anneal to the nucleotide tag, forming a loop between the annealed first and second tag-specific portions.

在具体实施方案中，例如在DNA测序中有用的，加标签的核苷酸序列另外包含在靶核苷酸序列和第一条形码核苷酸序列之间的引物结合位点。在此类实施方案的变化形式中，条形码引物的第一和第二标签特异性部分足够长以用作引物结合位点。为了帮助测序，这些结合位点的一个或多个、或优选地所有，是DNA测序引物的结合位点。在此类实施方案中，组合加标签的靶核苷酸序列可包含5'-第一标签特异性部分-第二条形码核苷酸序列-第二标签特异性部分-靶核苷酸序列-引物结合位点-第一条形码核苷酸序列-3'。In specific embodiments, such as those useful in DNA sequencing, the tagged nucleotide sequence further comprises a primer binding site between the target nucleotide sequence and the first barcode nucleotide sequence. In variations of such embodiments, the first and second tag-specific portions of the barcode primer are sufficiently long to serve as primer binding sites. To facilitate sequencing, one or more, or preferably all, of these binding sites are binding sites for DNA sequencing primers. In such embodiments, the combinatorially tagged target nucleotide sequence can comprise 5′-first tag-specific portion-second barcode nucleotide sequence-second tag-specific portion-target nucleotide sequence-primer binding site-first barcode nucleotide sequence-3′.

在一些实施方案中，有利地在加标签的靶核苷酸序列中包含一个或多个另外的核苷酸序列，例如，以帮助操作和/或鉴定。如此，加标签的靶核苷酸序列可包含第一另外的核苷酸序列，具有以下布置：5'-核苷酸标签-靶核苷酸序列-引物结合位点-第一条形码核苷酸序列-第一另外的核苷酸序列-3'。例如，在Illumina测序中，流动小室结合序列(例如，PE1和PE2)被掺入在待测序的DNA模板的任一末端。在本方法中，加标签的靶核苷酸序列可包含一个流动小室结合序列作为第一另外的核苷酸序列，且另一个流动小室结合序列可经由条形码引物被引入。参见，例如，图6。如此，本方法可采用包含第二另外的核苷酸序列并具有以下布置的条形码引物：5'-第二另外的核苷酸序列-第一标签特异性部分-第二条形码核苷酸序列-第二标签特异性部分-3'。在这种情形中，扩增产生组合加标签的靶核苷酸序列，包含5'-第二另外的核苷酸序列-第一标签特异性部分-第二条形码核苷酸序列-第二标签特异性部分-靶核苷酸序列-引物结合位点-第一条形码核苷酸序列-第一另外的核苷酸序列-3'。在这一实施方案的变化形式中，第一和/或第二另外的核苷酸序列包含引物结合位点。In some embodiments, it is advantageous to include one or more additional nucleotide sequences in the tagged target nucleotide sequence, for example, to aid in manipulation and/or identification. Thus, the tagged target nucleotide sequence can comprise a first additional nucleotide sequence having the following arrangement: 5'-nucleotide tag-target nucleotide sequence-primer binding site-first barcode nucleotide sequence-first additional nucleotide sequence-3'. For example, in Illumina sequencing, flow cell binding sequences (e.g., PE1 and PE2) are incorporated at either end of the DNA template to be sequenced. In the present method, the tagged target nucleotide sequence can comprise one flow cell binding sequence as the first additional nucleotide sequence, and another flow cell binding sequence can be introduced via a barcode primer. See, for example, FIG6 . Thus, the present method can employ a barcode primer comprising a second additional nucleotide sequence and having the following arrangement: 5'-second additional nucleotide sequence-first tag-specific portion-second barcode nucleotide sequence-second tag-specific portion-3'. In this case, amplification produces a combinatorially tagged target nucleotide sequence comprising 5′-second additional nucleotide sequence-first tag-specific portion-second barcode nucleotide sequence-second tag-specific portion-target nucleotide sequence-primer binding site-first barcode nucleotide sequence-first additional nucleotide sequence-3′. In variations of this embodiment, the first and/or second additional nucleotide sequence comprises a primer binding site.

靶核苷酸序列可通过任何方便的手段加标签，包括本文描述的基于引物的方法。在某些实施方案中，核苷酸标签包含转座子末端，其利用转座酶被掺入到加标签的靶核苷酸序列中。The target nucleotide sequence can be tagged by any convenient means, including the primer-based methods described herein. In certain embodiments, the nucleotide tag comprises a transposon end that is incorporated into the tagged target nucleotide sequence using a transposase.

掺入核酸序列的反应Reactions for incorporating nucleic acid sequences

可采用任何方法以掺入核酸序列到靶核酸中。在示意性实施方案中，采用PCR。当使用三个或更多个引物时，扩增通常进行至少三个循环以掺入第一和第二核苷酸标签以及条形码核苷酸序列。在多个实施方案中，扩增进行5、10、15、20、25、30、35、40、45或50个循环、或落入以这些值的任一个作为端点的范围中的任何数目的循环(例如5-10个循环)。在具体实施方案中，扩增进行足够数目的循环以将靶扩增子拷贝数跨靶和跨样品标准化(例如，15、20、25、30、35、40、45或50个循环、或落入以这些值的任一个作为端点的范围中的任何数目的循环)。In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification. In some embodiments, the target nucleic acid is amplified by amplification.

上述方法的特定实施方案提供大致上均一的扩增，产生多个靶扩增子，其中大多数扩增子以相对接近对多个靶扩增子计算的平均拷贝数的水平存在。如此，在多个实施方案中，靶扩增子的至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％以大于靶扩增子的平均拷贝数的50％和小于靶扩增子的平均拷贝数的2倍存在。Particular embodiments of the above methods provide for substantially uniform amplification, producing a plurality of target amplicons wherein a majority of the amplicons are present at a level relatively close to the average copy number calculated for the plurality of target amplicons. Thus, in various embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the target amplicons are present at greater than 50% and less than 2 times the average copy number of the target amplicons.

应用application

在示意性实施方案中，条形码核苷酸序列辨识特定样品。如此，例如，T个靶核酸的组可在S个样品的每个中扩增，其中S和T是整数，通常大于1。在此类实施方案中，扩增可对每个样品分别进行，其中相同的正向和反向引物组用于每个样品，且正向和反向引物组具有该组中所有引物共同的至少一个核苷酸标签。不同的条形码引物可用于每个样品，其中条形码引物具有不同的条形码核苷酸序列、但相同的可退火到共同的核苷酸标签的标签特异性部分。这个实施方案具有的益处是，减少对多个靶序列产生的扩增子中编码样品来源需要被合成的不同的引物的数目。可选地，不同的正向和反向引物组可用于每个样品，其中每个组具有不同于另一组中的引物的核苷酸标签，且不同的条形码引物用于每个样品，其中条形码引物具有不同的条形码核苷酸序列和不同的标签特异性部分。在任一种情形中，扩增从每个样品产生带有样品特异性条形码的T个扩增子的组。In an illustrative embodiment, a barcode nucleotide sequence identifies a specific sample. Thus, for example, a group of T target nucleic acids can be amplified in each of S samples, where S and T are integers, typically greater than 1. In such embodiments, amplification can be performed separately for each sample, wherein the same forward and reverse primer sets are used for each sample, and the forward and reverse primer sets have at least one nucleotide tag common to all primers in the set. Different barcode primers can be used for each sample, wherein the barcode primers have different barcode nucleotide sequences but the same tag-specific portion that can anneal to the common nucleotide tag. This embodiment has the benefit of reducing the number of different primers that need to be synthesized to encode the sample source in the amplicons generated for multiple target sequences. Alternatively, different forward and reverse primer sets can be used for each sample, wherein each set has a nucleotide tag different from the primers in the other set, and different barcode primers are used for each sample, wherein the barcode primers have different barcode nucleotide sequences and different tag-specific portions. In either case, amplification generates a group of T amplicons with sample-specific barcodes from each sample.

在其中相同的正向和反向引物组用于每个样品的实施方案中，每个靶的正向和反向引物可与样品分别地最初合并，且每个条形码引物可与其相应的样品最初合并。然后可将最初合并的正向和反向引物的小份加入到最初合并的样品和条形码引物的小份以产生S×T个扩增混合物。这些扩增混合物可在任何物品中形成，所述物品可经受适于扩增的条件。例如，在扩增之前，扩增混合物可在微流体装置的分别的区室中形成或被分配到微流体装置的分别的区室中。在示意性实施方案中，适合的微流体装置包括矩阵型微流体装置，诸如以下描述的那些。In embodiments where the same forward and reverse primer sets are used for each sample, the forward and reverse primers for each target can be initially merged with the sample separately, and each barcode primer can be initially merged with its corresponding sample. A small portion of the initially merged forward and reverse primers can then be added to the initially merged sample and a small portion of the barcode primers to produce S×T amplification mixtures. These amplification mixtures can be formed in any article that can be subjected to conditions suitable for amplification. For example, prior to amplification, the amplification mixtures can be formed in or distributed to separate compartments of a microfluidic device. In illustrative embodiments, suitable microfluidic devices include matrix-type microfluidic devices, such as those described below.

在某些实施方案中，在本文描述的任何方法中产生的靶扩增子可从扩增混合物回收。例如，适于允许回收每个反应区室的内容物的矩阵型微流体装置(参见以下)可用于扩增以产生靶扩增子。在这些实施方案的变化形式中，可对靶扩增子进行进一步的扩增和/或分析。在某些实施方案中，扩增混合物中产生的靶扩增子的量可在扩增期间定量，例如，通过定量实时PCR，或之后定量。In certain embodiments, the target amplicon produced in any of the methods described herein can be recovered from the amplification mixture. For example, a matrix-type microfluidic device (see below) suitable for allowing the recovery of the contents of each reaction compartment can be used for amplification to produce the target amplicon. In variations of these embodiments, the target amplicon can be further amplified and/or analyzed. In certain embodiments, the amount of the target amplicon produced in the amplification mixture can be quantified during amplification, for example, by quantitative real-time PCR, or quantified thereafter.

在可用于单颗粒分析的实施方案中，组合条形码化可用于编码反应体积的身份、以及因此为扩增产物的来源的颗粒的身份。在具体实施方案中，核酸扩增利用至少两个条形码序列进行，且条形码序列的组合编码为反应产物的来源的反应体积的身份(称为"组合条形码化")。当分别的反应体积处于矩阵型微流体装置的分别的区室中时方便地采用这些实施方案，例如，如从Fluidigm Corp.(South San Francisco,CA)可得的和以下描述的那些(参见"微流体装置")。每个分别的区室可包含辨识在其中进行编码反应的区室的行和列的条形码核苷酸序列的组合。如果回收反应体积并对其进行包括检测条形码组合的进一步分析(例如，通过DNA测序)，可将结果与特定区室关联，并从而与该区室中的特定颗粒关联。当在回收过程期间或之后合并分别的反应体积，从而合并("汇集")来自多个分别的反应体积的反应产物时，此类实施方案特别有用。在矩阵型微流体装置中，例如，可汇集来自行中的所有区室、列中的所有区室、或装置中的所有区室的反应产物。如果汇集行中的所有区室，行中的每列优选地具有独特的条形码组合。如果汇集列中的所有区室，列中的每行具有独特的条形码组合。如果汇集装置中的所有区室，装置中的每个区室具有独特的条形码组合。In embodiments that can be used for single particle analysis, combinatorial barcoding can be used to encode the identity of the reaction volume, and therefore the identity of the particle that is the source of the amplified product. In specific embodiments, nucleic acid amplification is performed using at least two barcode sequences, and the combination of the barcode sequences encodes the identity of the reaction volume that is the source of the reaction product (referred to as "combinatorial barcoding"). These embodiments are conveniently employed when the separate reaction volumes are in separate compartments of a matrix-type microfluidic device, for example, such as those available from Fluidigm Corp. (South San Francisco, CA) and described below (see "Microfluidic Devices"). Each separate compartment can contain a combination of barcode nucleotide sequences that identify the row and column of the compartment in which the encoding reaction was performed. If the reaction volumes are recovered and subjected to further analysis (e.g., by DNA sequencing) that includes detecting the barcode combination, the results can be associated with a specific compartment and, thereby, with a specific particle in that compartment. Such embodiments are particularly useful when the separate reaction volumes are combined during or after the recovery process, thereby combining ("pooling") the reaction products from multiple separate reaction volumes. In a matrix-type microfluidic device, for example, reaction products from all compartments in a row, all compartments in a column, or all compartments in a device can be pooled. If all compartments in a row are pooled, each column in the row preferably has a unique barcode combination. If all compartments in a column are pooled, each row in the column has a unique barcode combination. If all compartments in a device are pooled, each compartment in the device has a unique barcode combination.

条形码化和汇集反应混合物用于后续分析Barcoding and pooling of reaction mixtures for subsequent analysis

在其他实施方案中，条形码化和汇集策略用于在单独反应混合物中检测多个靶核酸，所述单独反应混合物可例如包含单独颗粒，诸如细胞。这一策略在以下实施例7中对基因表达的单细胞分析描述。In other embodiments, a barcoding and pooling strategy is used to detect multiple target nucleic acids in a single reaction mixture, which may, for example, contain individual particles, such as cells. This strategy is described below in Example 7 for single-cell analysis of gene expression.

在一个实施方案中，方法包括准备将在检验前被汇集的M个第一反应混合物，其中M是大于1的整数。每个反应混合物包含样品核酸；第一、正向引物，包含靶特异性部分；和第一、反向引物，包含靶特异性部分。第一、正向引物或第一、反向引物可另外包含条形码核苷酸序列，其中M个反应混合物的每一个中的每个条形码核苷酸序列是不同的。可选地，第一、正向引物或第一、反向引物另外包含核苷酸标签，且每个反应混合物另外包含至少一个条形码引物，条形码引物包含条形码核苷酸序列和核苷酸标签特异性部分，其中M个反应混合物的每一个中的每个条形码核苷酸序列是不同的。在这一实施方案中，条形码引物一般是比第一、正向和/或第一、反向引物过量的。对每个第一反应混合物进行第一反应以产生多个条形码化的靶核苷酸序列，各自包含靶核苷酸序列连接于条形码核苷酸序列。对于M个反应混合物的每一个，汇集条形码化的靶核苷酸序列以形成检验池。在这一检验池中，来自特定反应混合物的特定靶核苷酸序列被特定条形码核苷酸序列独特地辨识。使用独特的第二引物对对检验池或其一个或多个等份进行第二反应，其中每个第二引物对分别包含退火到靶核苷酸序列的第二、正向或反向引物；和退火到条形码核苷酸序列的第二、反向或正向引物。方法包括对每个独特的、第二引物对，确定反应产物是否存在于检验池或其等份中。对每个独特的、第二引物对，反应产物的存在指示特定第一反应混合物中特定靶核酸的存在。In one embodiment, the method includes preparing M first reaction mixtures to be pooled prior to testing, where M is an integer greater than 1. Each reaction mixture comprises a sample nucleic acid; a first forward primer comprising a target-specific portion; and a first reverse primer comprising a target-specific portion. The first forward primer or the first reverse primer may additionally comprise a barcode nucleotide sequence, wherein each barcode nucleotide sequence in each of the M reaction mixtures is different. Alternatively, the first forward primer or the first reverse primer additionally comprises a nucleotide tag, and each reaction mixture additionally comprises at least one barcode primer comprising a barcode nucleotide sequence and a nucleotide tag-specific portion, wherein each barcode nucleotide sequence in each of the M reaction mixtures is different. In this embodiment, the barcode primer is generally in excess of the first forward and/or first reverse primer. A first reaction is performed on each first reaction mixture to produce a plurality of barcoded target nucleotide sequences, each comprising a target nucleotide sequence linked to a barcode nucleotide sequence. For each of the M reaction mixtures, the barcoded target nucleotide sequences are pooled to form a test pool. In this test pool, a specific target nucleotide sequence from a specific reaction mixture is uniquely identified by a specific barcode nucleotide sequence. A second reaction is performed on the test pool or one or more aliquots thereof using a unique second primer pair, wherein each second primer pair comprises a second, forward or reverse primer that anneals to the target nucleotide sequence; and a second, reverse or forward primer that anneals to the barcode nucleotide sequence. The method includes determining, for each unique, second primer pair, whether a reaction product is present in the test pool or an aliquot thereof. For each unique, second primer pair, the presence of the reaction product indicates the presence of the specific target nucleic acid in the specific first reaction mixture.

在某些实施方案中，方法包括制备MxN个第一反应混合物，其中N是大于1的整数，且每个第一反应混合物包含对不同的靶核酸特异性的第一、正向和反向引物对。在第一反应后，制备各自包含M个第一反应混合物的N个检验池，其中检验池中每个条形码化的靶核苷酸序列包含不同的条形码核苷酸序列。第二反应在N个检验池的每一个中进行，每个检验池与每个其他的检验池是分开的。In certain embodiments, the method comprises preparing M x N first reaction mixtures, where N is an integer greater than 1, and each first reaction mixture comprises a first, forward, and reverse primer pair specific for a different target nucleic acid. Following the first reaction, N test pools are prepared, each comprising the M first reaction mixtures, wherein each barcoded target nucleotide sequence in the test pool comprises a different barcode nucleotide sequence. A second reaction is performed in each of the N test pools, each test pool being separate from every other test pool.

对于第一反应，可进行能够产生靶核苷酸序列连接于条形码核苷酸序列的任何反应。方便的第一反应包括扩增和连接。For the first reaction, any reaction that can produce a target nucleotide sequence linked to a barcode nucleotide sequence can be performed. A convenient first reaction includes amplification and ligation.

第二反应可以是依赖于条形码化的靶核苷酸序列的基于引物的检测的任何反应。可使用包括扩增和/或连接步骤，包括本文描述和/或本领域已知的那些的任一种的方法。例如，反应产物的存在可利用聚合酶链式反应(PCR)或连接酶链式反应(LCR)检测。在一些实施方案中，采用实时检测。The second reaction can be any reaction of the detection based on primers that depends on the target nucleotide sequence of barcoding.Can use and comprise amplification and/or connection step, comprise any method of those described herein and/or known in the art.For example, the presence of reaction product can utilize polymerase chain reaction (PCR) or ligase chain reaction (LCR) detection.In some embodiments, adopt real-time detection.

示例性的第二反应可采用LCR来检测具有以下结构的条形码化的靶核苷酸序列：5'-正向引物序列-靶核苷酸序列-反向引物序列-条形码核苷酸序列-3'。在这种情形中，一个引物可退火到反向引物序列，另一个引物可退火到邻近的条形码核苷酸序列，随后是连接、和重复的退火和连接循环。反向引物序列提供靶信息，且条形码核苷酸序列辨识池(其可，例如，代表特定样品中扩增的所有靶的池)。参见图8A。An exemplary second reaction can use LCR to detect a barcoded target nucleotide sequence having the following structure: 5'-forward primer sequence-target nucleotide sequence-reverse primer sequence-barcode nucleotide sequence-3'. In this case, one primer can anneal to the reverse primer sequence, and the other primer can anneal to the adjacent barcode nucleotide sequence, followed by ligation and repeated annealing and ligation cycles. The reverse primer sequence provides target information, and the barcode nucleotide sequence identifies a pool (which can, for example, represent the pool of all targets amplified in a particular sample). See Figure 8A.

示例性的第二反应可包括实时检测，例如，利用悬垂片核酸内切酶-连接酶链式反应。这一反应采用标记的探针和未标记的探针，其中探针对反应产物的同时杂交导致在标记的探针的5'末端形成悬垂片，且悬垂片的裂解产生信号。例如，悬垂片的裂解可分离荧光团与猝灭剂以产生信号。可采用示例性的实施方案来检测具有以下结构的反应产物：5'-正向引物序列-靶核苷酸序列-反向引物序列-条形码核苷酸序列-3'。在这种情形中，反应可采用退火到反向引物序列的未标记的探针和退火到邻近的条形码核苷酸序列的标记的探针。未标记的探针的3'末端的退火阻止标记的探针的5'末端的退火，形成悬垂片。这一5'悬垂片部分可用荧光团标记，且退火到条形码核苷酸序列的部分可带有猝灭剂，从而悬垂片被酶诸如5'悬垂片核酸内切酶的裂解释放悬垂片，藉以猝灭剂不再能够猝灭荧光团。参见图8B。An exemplary second reaction may include real-time detection, for example, using an overhanging piece endonuclease-ligase chain reaction. This reaction employs a labeled probe and an unlabeled probe, wherein simultaneous hybridization of the probe pairs to the reaction product results in the formation of an overhanging piece at the 5' end of the labeled probe, and the cleavage of the overhanging piece generates a signal. For example, the cleavage of the overhanging piece can separate the fluorophore from the quencher to generate a signal. An exemplary embodiment can be used to detect a reaction product having the following structure: 5'-forward primer sequence-target nucleotide sequence-reverse primer sequence-barcode nucleotide sequence-3'. In this case, the reaction can employ an unlabeled probe annealed to the reverse primer sequence and a labeled probe annealed to an adjacent barcode nucleotide sequence. Annealing of the 3' end of the unlabeled probe prevents annealing of the 5' end of the labeled probe, forming an overhanging piece. This 5' overhang portion can be labeled with a fluorophore, and the portion annealed to the barcode nucleotide sequence can carry a quencher, such that cleavage of the overhang by an enzyme such as a 5' overhang endonuclease releases the overhang, whereby the quencher can no longer quench the fluorophore. See Figure 8B.

有用的替代性实时检测方法，例如，用于检测通过LCR产生的扩增子，依赖于使用双链DNA结合染料来检测反应产物和LCR所用的引物之间的解链温度差异。解链温度分析包括在反应产物是大致上双链且在双链DNA结合染料存在下能够产生信号、但引物是大致上单链且不能产生信号的温度下检测。例如，对于检测具有以下结构的条形码化的靶核苷酸序列：5'-正向引物序列-靶核苷酸序列-反向引物序列-条形码核苷酸序列-3'，一个引物可退火到反向引物序列，另一个引物可退火到邻近的条形码核苷酸序列，随后是连接、和重复的退火和连接循环。参见图8C。连接的引物序列例如，R₁加BC₁与其互补物的长度比R₁或BC₁与其互补物的长度足够地长，从而在高温下，连接的引物序列是大致上双链的(即产生信号)，而未连接的引物序列是大致上单链的(即不产生信号)。在多个实施方案中，至少约10％、20％、30％、40％、50％、60％、70％、80％或90％的未连接的引物是单链的。在这些实施方案的每一个中，为双链的连接的引物的百分比可以是至少约10％、20％、30％、40％、50％、60％、70％、80％或90％。Useful alternative real-time detection methods, for example, for detecting amplicons produced by LCR, rely on the use of double-stranded DNA binding dyes to detect the difference in melting temperatures between the reaction products and the primers used for LCR. Melting temperature analysis includes detection at a temperature at which the reaction product is substantially double-stranded and can generate a signal in the presence of a double-stranded DNA binding dye, but the primers are substantially single-stranded and cannot generate a signal. For example, for detecting a barcoded target nucleotide sequence having the following structure: 5'-forward primer sequence-target nucleotide sequence-reverse primer sequence-barcode nucleotide sequence-3', one primer can anneal to the reverse primer sequence, and another primer can anneal to the adjacent barcode nucleotide sequence, followed by ligation, and repeated annealing and ligation cycles. See Figure 8C. The length of the ligated primer sequence, for example,_R1 plus_BC1 and its complement, is sufficiently longer than the length of_R1 or_BC1 and its complement, so that at high temperatures, the ligated primer sequence is substantially double-stranded (i.e., generates a signal), while the unligated primer sequence is substantially single-stranded (i.e., does not generate a signal). In a plurality of embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the unconnected primers are single-stranded. In each of these embodiments, the percentage of the primers that are double-stranded can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

在某些实施方案中，在微流体装置的分别的区室中准备第一反应混合物，分别的区室被布置为由行和列界定的阵列，例如，如从Fluidigm Corp.(South San Francisco,CA)可获得和以下描述的那些(参见"微流体装置")。例如，适于允许回收反应区室的内容物的矩阵型微流体装置(参见以下)可用于第一反应。这一方法对于准备各自包含M个第一反应混合物的N个检验池尤其方便。更具体地，第一反应在微流体装置的分别的区室中进行，其中分别的区室被布置为由行和列界定的阵列。N个检验池的每一个通过汇集装置的行或列中的第一反应混合物来获得。每个条形码化的靶核苷酸序列中的条形码核苷酸序列、连同检验池的身份，辨识为条形码化的靶核苷酸序列的来源的区室的行和列。在具体实施方案中，在微流体装置的分别的区室中准备第二反应混合物，微流体装置具有被布置为由行和列界定的阵列的分别的区室。例如，可在第一微流体装置的分别的区室中准备第一反应混合物以掺入条形码核苷酸序列(例如，Fluidigm Corporation的ACCESS ARRAY^TM IFC(Integrated Fluidic Circuit)或MA006 IFC)，在第二、不同的微流体装置的分别的区室中准备第二反应混合物以例如帮助检测(例如，Fluidigm Corporation的DYNAMIC ARRAY^TMIFC之一，利用PCR或RT-PCR，以双链DNA结合染料诸如EvaGreen用于检测)。In certain embodiments, the first reaction mixture is prepared in separate compartments of a microfluidic device, the separate compartments being arranged as an array defined by rows and columns, for example, such as those available from Fluidigm Corp. (South San Francisco, CA) and described below (see "Microfluidic Devices"). For example, a matrix-type microfluidic device (see below) suitable for allowing the recovery of the contents of the reaction compartments can be used for the first reaction. This method is particularly convenient for preparing N test cells, each containing M first reaction mixtures. More specifically, the first reaction is carried out in separate compartments of a microfluidic device, wherein the separate compartments are arranged as an array defined by rows and columns. Each of the N test cells is obtained by pooling the first reaction mixture in a row or column of the device. The barcode nucleotide sequence in each barcoded target nucleotide sequence, together with the identity of the test cell, identifies the row and column of the compartment that is the source of the barcoded target nucleotide sequence. In a specific embodiment, the second reaction mixture is prepared in separate compartments of a microfluidic device, the microfluidic device having separate compartments arranged as an array defined by rows and columns. For example, a first reaction mixture can be prepared in a separate compartment of a first microfluidic device to incorporate a barcode nucleotide sequence (e.g., Fluidigm Corporation's ACCESS ARRAY^™ IFC (Integrated Fluidic Circuit) or MA006 IFC), and a second reaction mixture can be prepared in a separate compartment of a second, different microfluidic device to, for example, facilitate detection (e.g., one of Fluidigm Corporation's DYNAMIC ARRAY^™ IFCs, using PCR or RT-PCR with a double-stranded DNA binding dye such as EvaGreen for detection).

在具体实施方案中，第一和/或第二反应的至少一个对单独颗粒诸如细胞进行。颗粒捕获和检验可如以下描述或如本领域已知的进行。Fluidigm Corporation的MA006 IFC充分适合于这一目的。颗粒在进行第一和/或第二反应时可以是大致上完整的，提供将与目的靶核酸接触的必需反应物。可选地，可在第一或第二反应之前破裂(disrupt)颗粒以帮助条形码化和/或后续分析。在一些实施方案中，在进行多个第一反应之前，用引发生物响应的剂处理颗粒。In a specific embodiment, at least one of the first and/or second reactions is performed on individual particles such as cells. Particle capture and testing can be performed as described below or as known in the art. Fluidigm Corporation's MA006 IFC is fully suitable for this purpose. The particles can be substantially intact when performing the first and/or second reactions, providing the necessary reactants to be contacted with the target nucleic acid of interest. Alternatively, the particles can be disrupted before the first or second reaction to help barcoding and/or subsequent analysis. In some embodiments, before performing multiple first reactions, the particles are treated with an agent that triggers a biological response.

后续分析Subsequent analysis

掺入核酸序列到靶核酸中的上述方法的任一种(包括上述的条形码化和汇集方法)可包括多种分析步骤的任一种，诸如确定第一反应混合物中至少一个靶核酸的量或确定第一反应混合物中一个或多个DNA分子的拷贝数。在其中加标签的或条形码化的靶核苷酸序列由PCR产生的某些实施方案中，例如，其中进行拷贝数确定的那些，有利地进行少于20个循环的PCR以保留不同的靶核苷酸序列的相对拷贝数。Any of the above methods for incorporating nucleic acid sequences into target nucleic acids (including the above-described barcoding and pooling methods) can include any of a variety of analytical steps, such as determining the amount of at least one target nucleic acid in the first reaction mixture or determining the copy number of one or more DNA molecules in the first reaction mixture. In certain embodiments in which the tagged or barcoded target nucleotide sequences are generated by PCR, for example, those in which copy number determination is performed, it is advantageous to perform fewer than 20 cycles of PCR to preserve the relative copy numbers of the different target nucleotide sequences.

上述方法的任一种可包括确定第一反应混合物中在一个或多个基因座处的基因型和/或确定第一反应混合物中多个基因座的单体型。单体型确定可例如通过以下进行：压缩(condensing)染色体和将染色体分配到第一反应混合物中以产生包含单个染色体的多个第一反应混合物。这一分配可例如如以下所述地对单颗粒分析进行(在这种情形中，被分析的"颗粒"是染色体)。可测序第一反应混合物中的多个基因座、以及因此必需在相同的染色体上的(therefore necessarily on the same chromosome)，以对那些基因座的提供单体型。Aforesaid method can comprise and determine the genotype at one or more loci and/or determine the haplotype of a plurality of loci in the first reaction mixture in the first reaction mixture.Haplotype is determined to for example be carried out by following: compression (condensing) chromosome and chromosome is assigned to a plurality of first reaction mixtures that comprise single chromosome to produce.This distribution can for example be carried out (in this case, analyzed " particle " is chromosome) to single particle analysis as described below).Can order-check a plurality of loci in the first reaction mixture and therefore must (therefore necessarily on the same chromosome) on identical chromosome, with haplotype to the provision of those loci.

在上述方法的任一种中，例如，其中进行RT-PCR时，可确定第一反应混合物中一个或多个RNA分子的表达水平。同DNA拷贝数确定一样，有利地进行少于20个循环的PCR以保留差异的相对拷贝数。In any of the above methods, for example, where RT-PCR is performed, the expression level of one or more RNA molecules in the first reaction mixture can be determined. As with DNA copy number determination, advantageously less than 20 cycles of PCR are performed to retain differential relative copy numbers.

无论第一反应混合物中的靶核酸是DNA或RNA，后续分析可包括确定从其产生的靶核苷酸序列的序列。Regardless of whether the target nucleic acid in the first reaction mixture is DNA or RNA, subsequent analysis can include determining the sequence of the target nucleotide sequence generated therefrom.

在一些实施方案中，本文描述的方法包括在每个第一反应混合物中进行多个反应，其中多个反应之一包括扩增以产生加标签的或条形码化的靶核苷酸序列，分析多个反应的结果，并将分析结果与每个第一反应混合物关联。这一关联可由如以上提及的加标签或条形码化靶核苷酸序列来帮助。例如，组合条形码化可用于编码关于来源反应混合物的信息。可选地，引物序列和条形码的组合可编码这一信息，如以上对于条形码化和汇集方法所讨论的。In some embodiments, the methods described herein include performing multiple reactions in each first reaction mixture, wherein one of the multiple reactions includes amplification to produce a tagged or barcoded target nucleotide sequence, analyzing the results of the multiple reactions, and associating the analysis results with each first reaction mixture. This association can be facilitated by the tagged or barcoded target nucleotide sequences as mentioned above. For example, combinatorial barcoding can be used to encode information about the source reaction mixture. Alternatively, a combination of primer sequences and barcodes can encode this information, as discussed above for the barcoding and pooling methods.

双向核酸测序Bidirectional nucleic acid sequencing

在具体实施方案中，本发明提供制备核酸用于双向DNA测序的方法，其帮助在单个读取测序运行中测序扩增产物的两个末端。此类方法在实施例9中示例。In a specific embodiment, the present invention provides a method for preparing nucleic acids for bidirectional DNA sequencing, which facilitates sequencing both ends of an amplification product in a single read sequencing run. Such a method is exemplified in Example 9.

待测序的DNA可以是任何类型的DNA。在具体实施方案中，DNA是来自生物体的基因组DNA或cDNA。在一些实施方案中，DNA可以是片段化的DNA。待测序的DNA可以是样品中RNA的代表，其中DNA通过例如逆转录或扩增RNA来获得。在某些实施方案中，DNA可以是DNA文库。The DNA to be sequenced can be any type of DNA. In specific embodiments, the DNA is genomic DNA or cDNA from an organism. In some embodiments, the DNA can be fragmented DNA. The DNA to be sequenced can be a representative of RNA in a sample, wherein the DNA is obtained by, for example, reverse transcription or amplification of RNA. In certain embodiments, the DNA can be a DNA library.

为了制备核酸用于根据本文描述的方法的双向DNA测序，利用内侧引物组扩增待测序的每个靶核酸，其中该组包含：To prepare nucleic acids for bidirectional DNA sequencing according to the methods described herein, each target nucleic acid to be sequenced is amplified using an inner primer set, wherein the set comprises:

内侧、正向引物，包含靶特异性部分和第一引物结合位点；an inner, forward primer containing a target-specific portion and a first primer binding site;

内侧、反向引物，包含靶特异性部分和第二引物结合位点，其中第一和第二引物结合位点是不同的。这些第一和第二引物结合位点起到双重功能，用作帮助添加进一步的核苷酸序列的核苷酸标签(如以下所述)，和在某些实施方案中，用作DNA测序引物可退火到的引物结合位点。在实施例9的具体实施方案中，第一和第二引物结合位点命名为"CS1"和"CS2"，代表"共有序列标签1"和"共有序列标签2"。在这一实施方案中，内侧引物的靶特异性部分命名为"TS-F"，代表"靶特异性正向"，和"TS-R"，代表"靶特异性反向"。The inner, reverse primer comprises a target-specific portion and a second primer binding site, wherein the first and second primer binding sites are different. These first and second primer binding sites serve a dual function, serving as nucleotide tags (as described below) that aid in the addition of further nucleotide sequences, and in certain embodiments, as primer binding sites to which DNA sequencing primers can anneal. In the specific embodiment of Example 9, the first and second primer binding sites are designated "CS1" and "CS2," standing for "consensus sequence tag 1" and "consensus sequence tag 2." In this embodiment, the target-specific portion of the inner primer is designated "TS-F," standing for "target-specific forward," and "TS-R," standing for "target-specific reverse."

扩增后，靶核苷酸序列变得被第一和第二引物结合位点加标签。将这些加标签的靶核苷酸序列退火到两组外侧引物，其退火到第一和第二引物结合位点。两组外侧引物包含：After amplification, the target nucleotide sequence becomes tagged by the first and second primer binding sites. These tagged target nucleotide sequences are annealed to two sets of outer primers, which anneal to the first and second primer binding sites. The two sets of outer primers contain:

第一外侧引物，其中该组包含：First outer primers, wherein the set comprises:

第一外侧、正向引物，包含对第一引物结合位点特异性的部分；和a first outer, forward primer comprising a portion specific for the first primer binding site; and

第一外侧、反向引物，包含条形码核苷酸序列和对第二引物结合位点特异性的部分；a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for the second primer binding site;

第二外侧引物组，其中该组包含：a second outer primer set, wherein the set comprises:

第二外侧、正向引物，包含条形码核苷酸序列和对第一引物结合位点特异性的部分；和a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and

第二外侧、反向引物，包含对第二引物结合位点特异性的部分。然后扩增产生两个靶扩增子，即：The second outer, reverse primer contains a portion specific for the second primer binding site. Amplification then produces two target amplicons, namely:

第一靶扩增子，包含5'-第一引物结合位点-靶核苷酸序列-第二引物结合位点-条形码核苷酸序列-3'；和A first target amplicon comprising 5'-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-3'; and

第二靶扩增子，包含5'-条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-3'。在具体实施方案中，两个靶扩增子的每个中的条形码核苷酸序列是相同的，且每个靶扩增子仅包含一个条形码核苷酸序列。在一些实施方案中，当扩增多于一个靶核酸时，产生的每对靶扩增子可具有相同的条形码序列，但不同的对可具有不同的条形码序列。在这种情形中，从不同的靶核酸产生的不同的靶扩增子之间的条形码序列将不同。如以上讨论的，例如，来自特定生物样品的不同的靶核酸组可用相同的组特异性序列(即，在组之间不同的序列)条形码化。在具体的实施方案中，组特异性条形码可以是样品特异性条形码，即，辨识靶扩增子所来自的样品的条形码。The second target amplicon comprises 5'-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-3'. In a specific embodiment, the barcode nucleotide sequence in each of the two target amplicons is identical, and each target amplicon comprises only one barcode nucleotide sequence. In some embodiments, when more than one target nucleic acid is amplified, each pair of target amplicons produced may have the same barcode sequence, but different pairs may have different barcode sequences. In this case, the barcode sequences between different target amplicons produced from different target nucleic acids will be different. As discussed above, for example, different target nucleic acid groups from a particular biological sample can be barcoded with the same group-specific sequence (i.e., a sequence that differs between the groups). In a specific embodiment, the group-specific barcode can be a sample-specific barcode, i.e., a barcode that identifies the sample from which the target amplicon comes.

在某些实施方案中，外侧引物各自另外包含另外的核苷酸序列，其中：In certain embodiments, the outer primers each further comprise an additional nucleotide sequence, wherein:

第一外侧、正向引物包含第一另外的核苷酸序列，且第一外侧、反向引物包含第二另外的核苷酸序列；和The first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and

第二外侧、正向引物包含第二另外的核苷酸序列，且第二外侧、反向引物包含第一另外的核苷酸序列，且第一和第二另外的核苷酸序列是不同的。在此类实施方案中，外侧引物扩增产生两个靶扩增子，即：The second outer, forward primer comprises a second additional nucleotide sequence, and the second outer, reverse primer comprises a first additional nucleotide sequence, and the first and second additional nucleotide sequences are different. In such embodiments, the outer primer amplification produces two target amplicons, namely:

第一靶扩增子，包含5'-第一另外的核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-条形码核苷酸序列-第二另外的核苷酸序列-3'；和a first target amplicon comprising 5′-first additional nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-second additional nucleotide sequence-3′; and

第二靶扩增子，包含5'-第二另外的核苷酸序列-条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一另外的核苷酸序列3'。(本领域技术人员理解，本文以这种方式描述的扩增子是对一条链描述的，互补链将具有这些核苷酸序列反向的5'至3'顺序。)The second target amplicon comprises 5'-second additional nucleotide sequence-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first additional nucleotide sequence 3'. (One skilled in the art will understand that the amplicon described herein in this manner is described for one strand, and the complementary strand will have the reversed 5' to 3' order of these nucleotide sequences.)

第一和/或第二另外的核苷酸序列还可包含引物结合位点。这一类型的示例性的引物构造描述在实施例9中，其中另外的核苷酸序列命名为"PE-1"和"PE-2"。这些序列是由Genome Analyzer(从Illumina,Inc.,San Diego,CA商购)使用的衔接子序列。条形码核苷酸序列命名为"BC"。利用这些引物的外侧引物扩增产生两个靶扩增子，即：The first and/or second additional nucleotide sequences may also contain primer binding sites. An exemplary primer configuration of this type is described in Example 9, wherein the additional nucleotide sequences are designated "PE-1" and "PE-2." These sequences are adapter sequences used by the Genome Analyzer (commercially available from Illumina, Inc., San Diego, CA). The barcode nucleotide sequence is designated "BC." Amplification using the outer primers of these primers produces two target amplicons, namely:

第一靶扩增子，包含5'-PE1-CS1-靶核苷酸序列-CS2-BC-PE2-3'；和A first target amplicon comprising 5'-PE1-CS1-target nucleotide sequence-CS2-BC-PE2-3'; and

第二靶扩增子，包含5'-PE2-BC-CS1-靶核苷酸序列-CS2-PE1-3'。在具体、示例性实施方案中，第一外侧引物组PE1-CS1和PE2-BC-CS2、和第二外侧引物组PE1-CS2和PE2-BC-CS1具有在实施例9的表1中所示的核苷酸序列。The second target amplicon comprises 5'-PE2-BC-CS1-target nucleotide sequence-CS2-PE1-3'. In a specific exemplary embodiment, the first outer primer set PE1-CS1 and PE2-BC-CS2, and the second outer primer set PE1-CS2 and PE2-BC-CS1 have the nucleotide sequences shown in Table 1 of Example 9.

内侧和外侧引物扩增可在单个扩增反应中进行。可选地，内侧引物扩增可在第一扩增反应中进行，且外侧引物扩增可在不同于第一的第二、扩增反应中进行。在某些实施方案中，第二扩增反应可在两个分别的第二扩增反应中进行：一个采用第一外侧引物组且另一个采用第二外侧引物组。参见实施例9、图2。在此类实施方案中，可汇集在每个分别的第二扩增反应中产生的靶扩增子用于进一步分析，诸如DNA测序。Inner and outer primer amplifications can be performed in a single amplification reaction. Alternatively, the inner primer amplification can be performed in a first amplification reaction, and the outer primer amplification can be performed in a second amplification reaction different from the first. In certain embodiments, the second amplification reaction can be performed in two separate second amplification reactions: one using the first outer primer set and the other using the second outer primer set. See Example 9, Figure 2. In such embodiments, the target amplicons produced in each separate second amplification reaction can be pooled for further analysis, such as DNA sequencing.

在许多实施方案中，上述方法将对多个靶核酸，诸如例如，DNA文库进行。在这种情形中，方法可用于对每个靶核酸产生包含两个类型扩增子(以上描述并在实施例9、图2中以"A"和"B"示例)的靶扩增子的池。一个类型的靶扩增子("A")帮助测序靶核酸的5'末端，另一个类型的靶扩增子("B")帮助测序靶核酸的3'末端。此外，每个靶扩增子包含条形码序列，在某些实施方案中，其在两个类型的靶扩增子的每种中是相同的。条形码核苷酸序列可编码关于靶核苷酸序列的信息，诸如产生其的反应的身份和/或靶核酸所来源自的样品的身份。如在以下更详细描述的，每个靶扩增子中的靶核苷酸序列和条形码核苷酸序列可利用任何适合的可得DNA测序方法容易地确定。在具体实施方案中，DNA测序方法是高通量测序方法，诸如桥式扩增(簇式产生(cluster generation))和由Illumina,Inc.,San Diego,CA商品化的测序方法。在某些实施方案中，例如，采用桥式扩增和测序的那些中，靶扩增子的平均长度是小于200个碱基、小于150个碱基或小于100个碱基。In many embodiments, the above method will be performed on multiple target nucleic acids, such as, for example, a DNA library. In this case, the method can be used to generate a pool of target amplicons comprising two types of amplicons (described above and exemplified as "A" and "B" in Example 9, FIG2 ) for each target nucleic acid. One type of target amplicon ("A") helps sequence the 5' end of the target nucleic acid, and the other type of target amplicon ("B") helps sequence the 3' end of the target nucleic acid. In addition, each target amplicon comprises a barcode sequence, which, in certain embodiments, is the same in each of the two types of target amplicons. The barcode nucleotide sequence can encode information about the target nucleotide sequence, such as the identity of the reaction that produced it and/or the identity of the sample from which the target nucleic acid was derived. As described in more detail below, the target nucleotide sequence and the barcode nucleotide sequence in each target amplicon can be easily determined using any suitable available DNA sequencing method. In a specific embodiment, the DNA sequencing method is a high-throughput sequencing method, such as bridge amplification (cluster generation) and a sequencing method commercialized by Illumina, Inc., San Diego, CA. In certain embodiments, eg, those employing bridge amplification and sequencing, the average length of the target amplicons is less than 200 bases, less than 150 bases, or less than 100 bases.

在桥式扩增和测序中，例如，将如本文所述产生的靶扩增子，经由第一和第二另外的核苷酸序列(例如，PE1和PE2)与固定的引物对的坪(a lawnof immobilized primerpairs)杂交。每个引物对中的一个固定的引物是可裂解的。进行第一链合成以产生双链分子。将这些变性，且洗掉用作第一链合成模板的最初杂交的靶扩增子链，留下固定的第一链。这些可翻转并与适合的邻近的引物杂交，形成桥。进行第二链合成以产生双链桥。将这些变性，每个桥产生两个固定的单链分子，其可再次与适合的固定的引物杂交。进行等温桥式扩增以产生多个双链桥。将双链桥变性，裂解并洗掉"反向"链，留下可用作DNA测序模板的固定的"正向"链的簇。In bridge amplification and sequencing, for example, the target amplicon produced as described herein is hybridized to a lawn of immobilized primer pairs via a first and a second additional nucleotide sequence (e.g., PE1 and PE2). One of the fixed primers in each primer pair is cleavable. First strand synthesis is performed to produce a double-stranded molecule. These are denatured, and the target amplicon strand initially hybridized as the first strand synthesis template is washed off, leaving a fixed first strand. These can be flipped and hybridized with suitable adjacent primers to form a bridge. Second strand synthesis is performed to produce a double-stranded bridge. These are denatured, and each bridge produces two fixed single-stranded molecules, which can again hybridize with suitable fixed primers. Isothermal bridge amplification is performed to produce multiple double-stranded bridges. The double-stranded bridge is denatured, cracked, and the "reverse" strand is washed off, leaving a cluster of fixed "forward" strands that can be used as a DNA sequencing template.

当对如本文所述产生的靶扩增子进行桥式扩增和测序时，可采用退火到第一和第二引物结合位点的引物(例如，CS1和CS2)来测序靶核苷酸序列或条形码核苷酸序列，二者都存在于从扩增子产生的固定的模板中。在某些实施方案中，在适于退火的条件下将适于测序靶核苷酸序列的引物对与固定的模板接触，随后DNA测序。读取这些序列后，可将测序产物变性和洗掉。然后可在适于退火的条件下将固定的模板与适于测序条形码核苷酸序列的引物对接触，随后DNA测序。这些测序反应的顺序不是关键并可以反转(即，可以先测序条形码核苷酸序列，随后测序靶核苷酸序列)。参见实施例9、图3。在某些实施方案中，引发测序条形码核苷酸序列的引物是引发测序靶核苷酸序列的引物的反向互补物。在具体、示例性实施方案中，用以引发测序靶核苷酸序列和条形码核苷酸序列的引物是CS1、CS2、CS1rc和CS2rc(表2，实施例9)。When the target amplicon produced as described herein is subjected to bridge amplification and sequencing, primers (e.g., CS1 and CS2) that anneal to the first and second primer binding sites can be used to sequence the target nucleotide sequence or the barcode nucleotide sequence, both of which are present in the fixed template generated from the amplicon. In certain embodiments, a primer pair suitable for sequencing the target nucleotide sequence is contacted with the fixed template under conditions suitable for annealing, followed by DNA sequencing. After reading these sequences, the sequencing products can be denatured and washed away. The fixed template can then be contacted with a primer pair suitable for sequencing the barcode nucleotide sequence under conditions suitable for annealing, followed by DNA sequencing. The order of these sequencing reactions is not critical and can be reversed (i.e., the barcode nucleotide sequence can be sequenced first, followed by sequencing the target nucleotide sequence). See Example 9, Figure 3. In certain embodiments, the primer that triggers the sequencing of the barcode nucleotide sequence is the reverse complement of the primer that triggers the sequencing of the target nucleotide sequence. In specific, exemplary embodiments, the primers used to trigger the sequencing of the target nucleotide sequence and the barcode nucleotide sequence are CS1, CS2, CS1rc, and CS2rc (Table 2, Example 9).

方便地，在相同的反应中对两个类型的靶扩增子进行桥式扩增和测序以允许同时测序来自每个类型靶扩增子的模板。参见实施例9、图3。这允许从5'末端(例如，在实施例9、图3中，通过测序来自A型扩增子的模板)和从3'末端(例如，在实施例9、图3中，通过测序来自B型扩增子的模板)同时测序每个靶核苷酸序列。在具体实施方案中，结合第一和第二引物结合位点并引发测序靶核苷酸序列的引物以大致上相等的浓度存在，从而产生来自每个靶核苷酸序列的5'和3'DNA序列信息。类似地，在某些实施方案中，结合第一和第二引物结合位点并引发测序条形码核苷酸序列的引物以大致上相等的浓度存在，从而产生来自每个模板类型的条形码序列(即，在实施例9、图3中，来源于A型扩增子或B型扩增子)。Conveniently, two types of target amplicons are bridge-amplified and sequenced in the same reaction to allow for simultaneous sequencing of templates from each type of target amplicons. See Example 9, Figure 3. This allows for simultaneous sequencing of each target nucleotide sequence from the 5' end (e.g., in Example 9, Figure 3, by sequencing the template from type A amplicon) and from the 3' end (e.g., in Example 9, Figure 3, by sequencing the template from type B amplicon). In a specific embodiment, primers that bind to the first and second primer binding sites and trigger sequencing target nucleotide sequences exist in roughly equal concentrations, thereby generating 5' and 3' DNA sequence information from each target nucleotide sequence. Similarly, in certain embodiments, primers that bind to the first and second primer binding sites and trigger sequencing barcode nucleotide sequences exist in roughly equal concentrations, thereby generating barcode sequences from each template type (i.e., in Example 9, Figure 3, derived from type A amplicon or type B amplicon).

当内侧扩增作为分别的反应进行时，尤其是当扩增多个靶核酸时，在微流体装置，诸如本文描述的或本领域已知的那些的任一个的分别的区室中进行单独反应(例如，每个反应扩增1、2、3、4、5或更多靶核酸)可以是方便的。如以下讨论的，适合的微流体装置可至少部分地从弹性体材料制造。When the inner amplification is performed as a separate reaction, especially when amplifying multiple target nucleic acids, it can be convenient to perform the separate reactions (e.g., each reaction amplifies 1, 2, 3, 4, 5, or more target nucleic acids) in separate compartments of a microfluidic device, such as any of those described herein or known in the art. As discussed below, suitable microfluidic devices can be fabricated at least in part from elastomeric materials.

在具体实施方案中，内侧或(内侧和外侧)扩增在设计为在进行扩增反应后帮助回收扩增产物的微流体装置中进行，诸如本文描述的(参见图2-9)并可从Fluidigm,Inc.,South San Francisco,CA获得的ACCESS ARRAY^TMIFC。在这一类型的示例性的装置中，可使用膨胀泵送(dilation pumping)以从微流体装置去除大致上所有反应产物，提供不同反应产物池之间的均匀性。如此，产生在体积和拷贝数方面是均匀的条形码化的反应产物的池是可能的。在多个实施方案中，体积和/或拷贝数均匀性是使得在体积和/或拷贝数方面，从装置回收的每个池的变率是小于约100％、小于约90％、小于约80％、小于约70％、小于约60％、小于约50％、小于约40％、小于约30％、小于约20％、小于约17％、或小于约15％、12％、10％、9％、8％、7％、6％、5％、4.5％、4％、3.5％、3％、2.5％、2％、1.5％、1％或0.5％。本领域技术人员理解，体积和/或拷贝数变率可落入以这些值的任一个为边界的任何范围内(例如，约2％至约7％)。在示例性实施方案中，从微流体装置回收的体积样品改变不大于大约10％。标准吸量误差是以5％至10％的数量级。如此，在体积方面观察到的变量主要可归因于吸量误差。与常规技术相比，利用本文描述的系统和方法减少了制备测序文库所需的时间和劳动。In a specific embodiment, the inner or (inner and outer) amplification is performed in a microfluidic device designed to facilitate recovery of amplification products after the amplification reaction is performed, such as the ACCESS ARRAY^™ IFC described herein (see Figures 2-9) and available from Fluidigm, Inc., South San Francisco, CA. In an exemplary device of this type, dilation pumping can be used to remove substantially all of the reaction products from the microfluidic device, providing uniformity between different reaction product pools. In this way, it is possible to generate pools of barcoded reaction products that are uniform in volume and copy number. In various embodiments, volume and/or copy number uniformity is such that in terms of volume and/or copy number, the variability of each pool recovered from the device is less than about 100%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 17%, or less than about 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1% or 0.5%. It will be appreciated by those skilled in the art that volume and/or copy number variability may fall within any range bounded by any of these values (e.g., about 2% to about 7%). In an exemplary embodiment, the volume sample recovered from the microfluidic device varies by no more than about 10%. The standard pipetting error is on the order of 5% to 10%. Thus, the variation observed in terms of volume is primarily attributable to pipetting error. Utilizing the systems and methods described herein reduces the time and labor required to prepare sequencing libraries compared to conventional techniques.

本领域技术人员将知晓可用于对多个不同的靶核酸、各自在分别的反应中进行本文所述的内侧(或内侧和外侧)扩增的其他装置和策略。例如，基于液滴的扩增充分适合于进行这一内侧扩增。参见，例如，2007年11月13日授权的Quake等人的美国专利号7,294,503，题为"Microfabricated crossflow devices and methods"，其通过引用全文并入本文，尤其是其形成和分析液滴的装置和方法的描述；2010年1月28日公布的Link等人的美国专利公布号20100022414，题为"Droplet libraries"，其通过引用全文并入本文，尤其是其形成和分析液滴的装置和方法的描述；和2011年1月6日公布的Miller等人的美国专利公布号20110000560，题为"Manipulation ofMicro fluidic Droplets"，其通过引用全文并入本文，尤其是其形成和分析液滴的装置和方法的描述。在具体实施方案中，内侧扩增在乳液的液滴中进行。Those skilled in the art will be aware of other devices and strategies that can be used to perform the inner (or inner and outer) amplification described herein on multiple different target nucleic acids, each in a separate reaction. For example, droplet-based amplification is well suited for performing this inner amplification. See, for example, U.S. Patent No. 7,294,503 to Quake et al., issued November 13, 2007, entitled “Microfabricated crossflow devices and methods,” which is incorporated herein by reference in its entirety, particularly for its description of devices and methods for forming and analyzing droplets; U.S. Patent Publication No. 20100022414 to Link et al., issued January 28, 2010, entitled “Droplet libraries,” which is incorporated herein by reference in its entirety, particularly for its description of devices and methods for forming and analyzing droplets; and U.S. Patent Publication No. 20110000560 to Miller et al., issued January 6, 2011, entitled “Manipulation of Micro fluidic Droplets,” which is incorporated herein by reference in its entirety, particularly for its description of devices and methods for forming and analyzing droplets. In specific embodiments, the internal amplification is performed in the droplets of the emulsion.

通过引物延伸编码和检测/定量等位基因Encoding and detection/quantification of alleles by primer extension

检测和评价核酸样品中特定靶核酸(例如，罕见突变)的比例的方法中可采用核酸编码。这一方法包括从样品中的第一和第二靶核酸产生第一和第二加标签的靶核苷酸序列。例如，方法可通过利用等位基因特异性扩增将等位基因特异性核苷酸标签引入所得的加标签的靶核苷酸序列来进行。然后对加标签的靶核苷酸序列进行利用对每个核苷酸标签特异性的引物的引物延伸反应。方法包括检测和/或定量指示第一引物的延伸的信号和指示第二引物的延伸的信号。给定引物的信号指示相应的靶核酸的存在、和/或相对量。这一方法可方便地在高通量(例如，下一代)DNA测序平台上进行以通过检测标签的存在，而不是通过确定每个分子的DNA序列检测例如样品中的已知突变。这一方法的益处是速度、灵敏度和准确。下一代测序中检查的大量克隆分子允许可靠地检测非常罕见的序列(例如，106个序列中小于1个)。而且，可以比PCR更准确地确定靶序列(例如，突变)的比例，因为下一代测序平台可获得非常高数目的读取。Nucleic acid encoding can be used in methods for detecting and evaluating the proportion of specific target nucleic acids (e.g., rare mutations) in a nucleic acid sample. This method includes generating first and second tagged target nucleotide sequences from first and second target nucleic acids in a sample. For example, the method can be performed by introducing allele-specific nucleotide tags into the resulting tagged target nucleotide sequences using allele-specific amplification. The tagged target nucleotide sequences are then subjected to a primer extension reaction using primers specific for each nucleotide tag. The method includes detecting and/or quantifying a signal indicating extension of the first primer and a signal indicating extension of the second primer. The signal for a given primer indicates the presence and/or relative amount of the corresponding target nucleic acid. This method can be conveniently performed on a high-throughput (e.g., next-generation) DNA sequencing platform to detect, for example, known mutations in a sample by detecting the presence of tags, rather than by determining the DNA sequence of each molecule. The benefits of this method are speed, sensitivity, and accuracy. The large number of clonal molecules examined in next-generation sequencing allows for the reliable detection of very rare sequences (e.g., less than 1 in 106 sequences). Moreover, the proportion of target sequences (e.g., mutations) can be determined more accurately than with PCR because next-generation sequencing platforms can obtain very high numbers of reads.

为了帮助DNA测序平台上的引物延伸，可将用于例如高通量DNA测序的衔接子引入第一和第二加标签的靶核苷酸序列。在具体实施方案中，将衔接子引入到加标签的靶核苷酸序列分子的每个末端。这些衔接子可方便地在一个反应中与核苷酸标签一起引入。To facilitate primer extension on a DNA sequencing platform, adapters used, for example, in high-throughput DNA sequencing can be introduced into the first and second tagged target nucleotide sequences. In a specific embodiment, an adapter is introduced to each end of the tagged target nucleotide sequence molecules. These adapters can be conveniently introduced together with the nucleotide tags in a single reaction.

核苷酸标签和/或DNA测序衔接子可利用任何适合的方法诸如例如，扩增或连接引入靶核苷酸序列。例如，可通过分别用第一和第二引物对扩增第一和第二靶核酸产生第一和第二加标签的靶核苷酸序列。第一引物对中的至少一个引物包含第一核苷酸标签且第二引物对中的至少一个引物包含第二核苷酸标签。当在相同的反应中引入DNA测序衔接子时，每个引物对中的一个引物包含5'-(DNA测序衔接子)-(核苷酸标签)-(靶特异性部分)-3'，且每个引物对中的另一个引物包含5'-(DNA测序衔接子)-(靶特异性部分)-3'。Nucleotide tags and/or DNA sequencing adapters can be introduced into the target nucleotide sequence using any suitable method, such as, for example, amplification or ligation. For example, first and second tagged target nucleotide sequences can be generated by amplifying first and second target nucleic acids using first and second primer pairs, respectively. At least one primer in the first primer pair comprises a first nucleotide tag and at least one primer in the second primer pair comprises a second nucleotide tag. When the DNA sequencing adapters are introduced in the same reaction, one primer in each primer pair comprises 5'-(DNA sequencing adapter)-(nucleotide tag)-(target-specific portion)-3' and the other primer in each primer pair comprises 5'-(DNA sequencing adapter)-(target-specific portion)-3'.

许多高通量DNA测序技术包括在DNA测序之前的扩增步骤。因此，在一些实施方案中，在DNA测序平台上引物延伸之前进一步扩增加标签的靶核苷酸序列。例如，可进行乳液扩增或桥式扩增。乳液PCR(emPCR)将单独的DNA分子连同引物涂覆的珠一起分离到位于油相中的水滴中。PCR生成了DNA分子的多个拷贝，这些DNA分子连接到珠上的引物上，紧接着进行固定用于后续测序。emPCR被用于由Marguilis等人(由454 Life Sciences,Branford,CT商品化)、Shendure以及Porreca等人(在本文称为“454测序”，也称为“聚合酶克隆测序(polony sequencing)”)以及SOLiD测序(Life Technologies,Foster City,CA)提供的方法中。参见M.Margulies,等人.(2005)"Genome sequencing in microfabricated high-density picolitre reactors"Nature437:376-380；J.Shendure,等人.(2005)"AccurateMultiplex Polony Sequencing of an Evolved Bacterial Genome"Science 309(5741):1728-1732。还可以通过“桥式PCR”进行体外克隆扩增，其中当引物附加到固体表面上时，片段被扩增。Braslavsky等人开发了一种单分子方法(由Helicos Biosciences Corp.,Cambridge,MA商品化)，该方法省略了这个扩增步骤，直接地将DNA分子固定到表面上。I.Braslavsky,等人.(2003)"Sequence information can be obtained from single DNAmolecules"Proceedings of the National Academy of Sciences of the UnitedStates of America 100:3960-3964。Many high-throughput DNA sequencing technologies include an amplification step before DNA sequencing. Therefore, in some embodiments, the target nucleotide sequence of the tag is further amplified before primer extension on the DNA sequencing platform. For example, emulsion amplification or bridge amplification can be performed. Emulsion PCR (emPCR) separates the individual DNA molecules together with the primer-coated beads into a water droplet in an oil phase. PCR generates multiple copies of the DNA molecule, which are connected to the primers on the beads and then fixed for subsequent sequencing. emPCR is used by the people such as Marguilis (commercialized by 454 Life Sciences, Branford, CT), the people such as Shendure and Porreca (referred to herein as "454 sequencing", also referred to as "polymerase clone sequencing (polony sequencing)") and SOLiD sequencing (Life Technologies, Foster City, CA) in the method provided. See M. Margulies, et al. (2005) "Genome sequencing in microfabricated high-density picolitre reactors" Nature 437:376-380; J. Shendure, et al. (2005) "Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome" Science 309(5741):1728-1732. In vitro clonal amplification can also be performed by "bridge PCR," in which fragments are amplified when primers are attached to a solid surface. Braslavsky et al. developed a single-molecule method (commercialized by Helicos Biosciences Corp., Cambridge, MA) that omits this amplification step and directly immobilizes the DNA molecules to the surface. I. Braslavsky, et al. (2003) "Sequence information can be obtained from single DNA molecules" Proceedings of the National Academy of Sciences of the United States of America 100:3960-3964.

被物理结合到表面上的DNA分子可以被平行地测序。“通过合成测序”，像染料终止电泳测序一样，使用DNA聚合酶来确定碱基序列。“焦磷酸测序”使用DNA聚合作用，一次加入一个核苷酸并且通过附加的焦磷酸盐的释放所发出的光对被加入到给定位置的核苷酸的数量进行检测和定量(由454 Life Sciences,Branford,CT商品化)。参见M.Ronaghi,等人.(1996)."Real-time DNA sequencing using detection of pyrophosphate release"Analytical Biochemistry 242:84-89。可逆的终止剂方法(由Illumina,Inc.,San Diego,CA和Helicos Biosciences Corp.,Cambridge,MA商品化)通过反复去除封闭基团以允许聚合另一个核苷酸使用染料终止剂的可逆形式，一次加入一个核苷酸，并且实时地对各位置上的荧光进行检测。DNA molecules physically bound to a surface can be sequenced in parallel. "Sequencing by synthesis," like dye-terminated electrophoretic sequencing, uses DNA polymerase to determine the base sequence. "Pyrophosphate sequencing" uses DNA polymerization to add one nucleotide at a time and detect and quantify the number of nucleotides added to a given position by light emitted by the release of the attached pyrophosphate (commercialized by 454 Life Sciences, Branford, CT). See M. Ronaghi, et al. (1996). "Real-time DNA sequencing using detection of pyrophosphate release" Analytical Biochemistry 242:84-89. Reversible terminator methods (commercialized by Illumina, Inc., San Diego, CA and Helicos Biosciences Corp., Cambridge, MA) use a reversible form of a dye terminator by repeatedly removing a blocking group to allow polymerization of another nucleotide, adding one nucleotide at a time, and detecting fluorescence at each position in real time.

在通过引物延伸的检测方法的一个实施方案中，其可方便地在454测序平台上进行，第一和第二引物延伸反应在引物延伸的至少两个循环中顺序地进行。具体地，引物延伸的第一循环利用退火到第一核苷酸标签的第一引物进行，引物延伸的第二循环利用退火到第二核苷酸标签的第二引物进行。在每个引物延伸循环中提供所有脱氧核苷三磷酸(dNTP)。任何dNTP向DNA分子的掺入产生可检测信号。在第一循环中检测的信号指示核酸样品中第一靶核酸的存在，而在第二循环中检测的信号指示核酸样品中第二靶核酸的存在。如此，可用仅单个循环的测序平台检测每个靶核酸(例如，突变)。In one embodiment of the detection method by primer extension, it can be easily carried out on 454 sequencing platforms, and the first and second primer extension reactions are carried out sequentially in at least two cycles of primer extension. Specifically, the first cycle of primer extension is carried out using a first primer annealed to a first nucleotide tag, and the second cycle of primer extension is carried out using a second primer annealed to a second nucleotide tag. All deoxynucleoside triphosphates (dNTPs) are provided in each primer extension cycle. Any dNTP produces a detectable signal to the incorporation of DNA molecules. The presence of the first target nucleic acid in the signal indication nucleic acid sample detected in the first cycle, and the presence of the second target nucleic acid in the signal indication nucleic acid sample detected in the second cycle. In this way, each target nucleic acid (for example, a mutation) can be detected on a sequencing platform with only a single cycle.

因为检测的信号是与靶核酸的拷贝数成比例的，信号还可用于评价样品中靶核酸的量。具体地，信号可用于确定两个或多个靶核酸相对彼此的量。Because the detected signal is proportional to the copy number of the target nucleic acid, the signal can also be used to assess the amount of the target nucleic acid in the sample. Specifically, the signal can be used to determine the amount of two or more target nucleic acids relative to each other.

在使用454测序平台检测野生型和突变体靶核酸的示例性的实施方案中，用待检测的野生型和每个突变体的特异性标签准备等位基因特异性PCR反应。如图31所示的，正向引物具有454衔接子和等位基因特异性标签(用不同的阴影辨识)。衔接子是在标签的5'，标签是在引物的等位基因特异性部分的5'。反向引物包含靶特异性部分5'的454衔接子。如图31所示的，仅需要一个反向引物来检测单核苷酸多态性。在这一实例中，在单个PCR反应中进行两个等位基因特异性PCR反应，而这不是该方法必需的。PCR反应产生对454珠乳液PCR准备的加标签的靶核苷酸序列。可省略乳液PCR步骤，例如，通过直接退火加标签的靶核苷酸序列到预负载有等位基因特异性寡核苷酸的珠(即，每个单独珠带有仅一个类型的寡核苷酸)。在任一种情形中，单独珠将带有仅一个类型的加标签的靶核苷酸。将珠上样到454测序仪。第一454循环流动例如结合野生型标签的引物和所有四种dNTP。随着这一引物延伸，掺入多个核苷酸，提供非常牢固的信号，但仅在包含野生型珠的孔中。第二454循环流动结合突变体标签的引物和所有四种dNTP，仅在包含突变体珠的孔中提供信号。In an exemplary embodiment of using a 454 sequencing platform to detect wild-type and mutant target nucleic acids, allele-specific PCR reactions are prepared with specific tags for the wild-type and each mutant to be detected. As shown in Figure 31, the forward primer has a 454 adapter and an allele-specific tag (identified by different shading). The adapter is 5' of the tag, and the tag is 5' of the allele-specific portion of the primer. The reverse primer contains a 454 adapter 5' of the target-specific portion. As shown in Figure 31, only one reverse primer is needed to detect single nucleotide polymorphisms. In this example, two allele-specific PCR reactions are performed in a single PCR reaction, which is not required for this method. The PCR reaction produces a tagged target nucleotide sequence prepared for 454 bead emulsion PCR. The emulsion PCR step can be omitted, for example, by directly annealing the tagged target nucleotide sequence to beads preloaded with allele-specific oligonucleotides (i.e., each individual bead carries only one type of oligonucleotide). In either case, the individual beads will carry only one type of tagged target nucleotide. The beads are loaded onto the 454 sequencer. The first 454-cycle flow, for example, involves a primer that binds to the wild-type tag and all four dNTPs. As this primer is extended, multiple nucleotides are incorporated, providing a very strong signal, but only in wells containing wild-type beads. The second 454-cycle flow involves a primer that binds to the mutant tag and all four dNTPs, providing a signal only in wells containing mutant beads.

在通过引物延伸的检测方法的另一实施方案中，其可在SOLiD测序平台上方便地进行，通过寡核苷酸连接和检测进行第一和第二引物延伸反应。在这一实施方案中，标记的二碱基寡核苷酸对第一和/或第二引物的连接产生可检测信号，且对特定引物检测的总信号指示核酸样品中相应的靶核酸的存在、和/或相对量。在这一实施方案的变化形式中，标记的二碱基寡核苷酸对第一引物的连接与标记的二碱基寡核苷酸对第二引物的连接产生相同的可检测信号，且第一和第二引物延伸反应分别进行，例如，在同时或顺序的循环中。在另一变化形式中，标记的二碱基寡核苷酸对第一引物的连接与标记的二碱基寡核苷酸对第二引物的连接产生不同的可检测信号。不同的信号的使用允许第一和第二引物延伸反应在一个反应混合物中同时进行。该方法中可采用任何类型的可检测信号，但通常采用荧光信号，例如，用于SOLiD测序的。In another embodiment of the detection method by primer extension, it can be carried out easily on the SOLiD order-checking platform, connects and detects and carries out the first and second primer extension reactions by oligonucleotide.In this embodiment, the dibase oligonucleotide of mark produces detectable signal to the connection of the first and/or second primer, and the existence and/or relative amount of corresponding target nucleic acid in the total signal indication nucleic acid sample that specific primer is detected.In the variation of this embodiment, the dibase oligonucleotide of mark produces identical detectable signal to the connection of the first primer and the dibase oligonucleotide of mark to the connection of the second primer, and the first and second primer extension reactions are carried out respectively, for example, in the circulation of simultaneously or sequence.In another variation, the dibase oligonucleotide of mark produces different detectable signals to the connection of the first primer and the dibase oligonucleotide of mark to the connection of the second primer.The use of different signals allows the first and second primer extension reactions to carry out simultaneously in a reaction mixture.Can adopt any type of detectable signal in the method, but adopt fluorescent signal usually, for example, for SOLiD order-checking.

为如以上描述的SOLiD测序平台上引物延伸准备包含例如等位基因特异性标签和适合的DNA测序衔接子的加标签的靶核苷酸序列。可进行乳液PCR，而这一步骤不是严格必需的。如以上关于454测序描述的，产生附加于珠的加标签的靶核苷酸序列的克隆群的任何方法可用于产生适于在SOLiD测序平台上引物延伸的加标签的靶核苷酸序列。Tagged target nucleotide sequences comprising, for example, allele-specific tags and suitable DNA sequencing adapters are prepared for primer extension on the SOLiD sequencing platform as described above. Emulsion PCR can be performed, although this step is not strictly necessary. Any method that produces clonal populations of tagged target nucleotide sequences attached to beads, as described above for 454 sequencing, can be used to generate tagged target nucleotide sequences suitable for primer extension on the SOLiD sequencing platform.

在通过引物延伸的检测方法的又另一实施方案中，其可方便地在Illumina测序平台上进行，第一和第二引物延伸反应包括通过合成测序。在这一实施方案中，每个脱氧核苷三磷酸用不同的、碱基特异性标记物标记，且脱氧核苷三磷酸向DNA分子的掺入产生碱基特异性可检测信号。对特定引物检测的总信号指示核酸样品中相应的靶核酸的存在和/或相对量。在这一实施方案的变化形式中，第一引物的延伸与第二引物的延伸产生相同的可检测信号，且第一和第二引物延伸反应分别进行，例如，在同时或顺序的循环中。在另一变化形式中，第一引物的延伸与第二引物的延伸产生不同的可检测信号。不同的信号的使用允许第一和第二引物延伸反应在一个反应混合物中同时进行。该方法中可采用任何类型的可检测信号，但通常采用荧光信号，例如，用于Illumina测序的。为如以上描述的Illumina测序平台上引物延伸准备包含等位基因特异性标签和适合的DNA测序衔接子的加标签的靶核苷酸序列。对于在Illumina测序平台上的引物延伸，在DNA测序之前，通常通过桥式PCR进一步扩增加标签的靶核苷酸序列。In another embodiment of the detection method by primer extension, it can be conveniently performed on the Illumina sequencing platform, and the first and second primer extension reactions include sequencing by synthesis. In this embodiment, each deoxynucleoside triphosphate is labeled with a different, base-specific marker, and the incorporation of the deoxynucleoside triphosphate into the DNA molecule produces a base-specific detectable signal. The total signal detected for a specific primer indicates the presence and/or relative amount of the corresponding target nucleic acid in the nucleic acid sample. In a variation of this embodiment, the extension of the first primer and the extension of the second primer produce the same detectable signal, and the first and second primer extension reactions are performed separately, for example, in simultaneous or sequential cycles. In another variation, the extension of the first primer and the extension of the second primer produce different detectable signals. The use of different signals allows the first and second primer extension reactions to be performed simultaneously in a reaction mixture. Any type of detectable signal can be used in this method, but fluorescent signals are generally used, for example, for Illumina sequencing. For primer extension on the Illumina sequencing platform as described above, a tagged target nucleotide sequence containing an allele-specific tag and a suitable DNA sequencing adapter is prepared. For primer extension on the Illumina sequencing platform, the tagged target nucleotide sequence is typically further amplified by bridge PCR prior to DNA sequencing.

在以上所述的具体的通过引物延伸的检测实施方案中，以及在该方法的一些其他实现中，扩增产生在或变得位于离散反应位点的加标签的靶核苷酸序列的克隆群。包含第一核苷酸标签的反应位点的数目相对于包含第二核苷酸标签的反应位点的数目指示样品中第一靶核酸相对于第二靶核酸的量。在这一类型的特定实施方案中，方法可包括检测和比较来自包含第一核苷酸标签的所有反应位点的总信号与来自包含第二核苷酸标签的所有反应位点的总信号。可选地或另外，方法可包括检测和比较包含第一核苷酸标签的反应位点的数目与包含第二核苷酸标签的反应位点的数目。在任一种情形中，比较可包括比较两个值的任何常规手段，诸如，例如，确定比例。In the specific detection embodiment by primer extension described above, as well as in some other implementations of the method, amplification produces or becomes located at discrete reaction sites of tagged target nucleotide sequences. The number of reaction sites comprising a first nucleotide tag relative to the number of reaction sites comprising a second nucleotide tag indicates the amount of the first target nucleic acid relative to the second target nucleic acid in the sample. In specific embodiments of this type, the method may include detecting and comparing the total signal from all reaction sites comprising the first nucleotide tag with the total signal from all reaction sites comprising the second nucleotide tag. Alternatively or in addition, the method may include detecting and comparing the number of reaction sites comprising the first nucleotide tag with the number of reaction sites comprising the second nucleotide tag. In either case, the comparison may include any conventional means of comparing two values, such as, for example, determining a ratio.

用于该方法的适合的、可区分的核苷酸标签的选择在本领域技术人员的能力范围内。在某些实施方案中，第一核苷酸标签可包含第一核苷酸的均聚物(例如，多聚-A)，而第二核苷酸标签可包含第二、不同的核苷酸的均聚物(例如，多聚-G)。The selection of suitable, distinguishable nucleotide tags for use in the method is within the capabilities of those skilled in the art. In certain embodiments, the first nucleotide tag may comprise a homopolymer of a first nucleotide (e.g., poly-A), and the second nucleotide tag may comprise a homopolymer of a second, different nucleotide (e.g., poly-G).

尽管通过引物延伸的检测方法在以上是对分析两个靶核酸描述的，该方法涵盖分析三个或更多个靶核酸，其每一个用不同的核苷酸标签加标签。对所得的加标签的靶核苷酸序列进行三个或更多个引物延伸反应，各自利用退火到不同核苷酸标签的引物，且对每个引物的延伸检测和/或定量信号。在具体实施方案中，两个或更多个加标签的靶核苷酸序列包括不同的条形码，如以上所述的，条形码可编码关于加标签的靶核苷酸序列的信息，例如，样品或反应混合物。Although the detection method by primer extension is described above for analyzing two target nucleic acids, the method encompasses analyzing three or more target nucleic acids, each tagged with a different nucleotide tag. The resulting tagged target nucleotide sequences are subjected to three or more primer extension reactions, each utilizing a primer that anneals to a different nucleotide tag, and the signal from each primer extension is detected and/or quantified. In specific embodiments, the two or more tagged target nucleotide sequences include different barcodes, which, as described above, can encode information about the tagged target nucleotide sequences, e.g., a sample or reaction mixture.

如果需要，以上通过引物延伸的检测方法可以多路进行。例如，在某些实施方案中，通过掺入一个或多个条形码到核苷酸标签中，其中条形码编码样品身份，可在一个或多个引物延伸反应中一起分析多个样品。对引物延伸反应，可采用为等位基因和条形码二者特异性的引物，或可选地，条形码可优选地邻近引物所退火到的核苷酸标签，且引物延伸反应可以是DNA测序反应，其仅需要检测条形码的序列。在前者实施方案中，引物延伸将指示来自特定样品的等位基因的存在，而在后者实施方案中，引物延伸将指示等位基因的存在，且条形码核苷酸序列将辨识样品。If desired, the above detection method by primer extension can be multiplexed. For example, in certain embodiments, multiple samples can be analyzed together in one or more primer extension reactions by incorporating one or more barcodes into the nucleotide tags, where the barcodes encode the sample identity. For the primer extension reactions, primers specific for both the allele and the barcode can be employed, or alternatively, the barcodes can be preferably adjacent to the nucleotide tags to which the primers anneal, and the primer extension reactions can be DNA sequencing reactions, which only require detection of the sequence of the barcodes. In the former embodiment, primer extension will indicate the presence of the allele from a particular sample, while in the latter embodiment, primer extension will indicate the presence of the allele, and the barcode nucleotide sequence will identify the sample.

单颗粒分析应用Single Particle Analysis Applications

掺入核酸序列到单颗粒中Incorporation of nucleic acid sequences into single particles

在某些实施方案中，掺入核酸序列到靶核酸中的上述方法(包括上述条形码化和汇集方法)在检验颗粒群体中的单颗粒的上下文中使用。一般，将核酸序列引入与颗粒缔合(associated with)或包含在颗粒中的靶核酸。如此，上述第一反应在包含单独颗粒的反应体积中进行。可开发将单颗粒分析结果与检验的每个颗粒关联的能力，其中例如，将两个或更多个参数与表型关联。测量的两个或更多个参数可以是不同类型的参数，例如，RNA表达水平和核苷酸序列。本文描述的单细胞分析方法的另外的应用在以下描述。In certain embodiments, the above-mentioned methods (including the above-mentioned barcoding and pooling methods) of incorporating nucleic acid sequences into target nucleic acids are used in the context of testing single particles in a population of particles. Generally, the nucleic acid sequence is introduced into a target nucleic acid associated with or contained in a particle. Thus, the above-mentioned first reaction is carried out in a reaction volume containing a single particle. The ability to associate the results of a single particle analysis with each particle tested can be developed, wherein, for example, two or more parameters are associated with a phenotype. The two or more parameters measured can be different types of parameters, for example, RNA expression levels and nucleotide sequences. Additional applications of the single cell analysis methods described herein are described below.

单颗粒分析包括在分别的反应体积中捕获群体的颗粒以产生各包含仅一个颗粒的多个分别的反应体积。包含颗粒的分别的反应体积可在液滴中、在乳液中、在容器中、在微量滴定板的孔中、或在矩阵型微流体装置的区室形成。在示意性实施方案中，分别的反应体积在微流体装置的单独区室中存在，所述微流体装置诸如例如，本文描述的那些的任一种。还参见，2004年11月18日公布的Daridon等人的美国专利公布号2004/0229349，其通过引用全文并入本文，尤其是其微流体颗粒分析系统的描述。Single particle analysis includes capturing particles of a population in separate reaction volumes to produce multiple separate reaction volumes each containing only one particle. The separate reaction volumes containing particles can be formed in droplets, in emulsions, in containers, in wells of microtiter plates, or in compartments of matrix-type microfluidic devices. In illustrative embodiments, the separate reaction volumes are present in separate compartments of a microfluidic device, such as, for example, any of those described herein. See also, U.S. Patent Publication No. 2004/0229349 to Daridon et al., published November 18, 2004, which is incorporated herein by reference in its entirety, particularly for its description of microfluidic particle analysis systems.

在某些实施方案中，通过以下检验参数：在每个分别的反应体积中进行反应诸如核酸扩增以产生一个或多个反应产物，分析反应产物以获得结果，随后将结果与颗粒关联并输入数据组。颗粒可被捕获在分别的反应体积中，然后与一个或多个试剂接触用于进行一个或多个反应。可选地或此外，颗粒可与一个或多个此类试剂接触，可将反应混合物分配到分别的反应体积中。在多个实施方案中，至少2、3、4、5、6、7、8、9、或10或更多个反应在每个分别的反应体积中进行。反应产物的分析可在分别的反应体积中进行。在一些实施方案中，然而，为了后续分析或其他目的，回收分别的反应体积的内容物是有利的。例如，如果核酸扩增在分别的反应体积中进行，可以期望回收内容物用于后续分析，例如，通过PCR和/或核酸测序。分别的反应体积的内容物可分别分析，并将结果与最初反应体积中存在的颗粒关联。可选地，可在反应产物中编码颗粒/反应体积身份，例如，如以上对多引物核酸扩增方法讨论的。另外，可合并这两种策略，从而编码分别的反应体积的组，使得组中的每个反应体积是独特地可辨识的，然后汇集，并分别分析每个池，如由上述条形码化和汇集方法示例的。In certain embodiments, the following assay parameters are used: a reaction, such as nucleic acid amplification, is performed in each separate reaction volume to produce one or more reaction products, the reaction products are analyzed to obtain results, and the results are subsequently associated with the particles and entered into a data set. The particles can be captured in separate reaction volumes and then contacted with one or more reagents for performing one or more reactions. Alternatively or in addition, the particles can be contacted with one or more such reagents, and the reaction mixture can be distributed among the separate reaction volumes. In various embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more reactions are performed in each separate reaction volume. Analysis of the reaction products can be performed in separate reaction volumes. In some embodiments, however, it is advantageous to recover the contents of the separate reaction volumes for subsequent analysis or other purposes. For example, if nucleic acid amplification is performed in separate reaction volumes, it may be desirable to recover the contents for subsequent analysis, e.g., by PCR and/or nucleic acid sequencing. The contents of the separate reaction volumes can be analyzed separately, and the results associated with the particles present in the initial reaction volume. Alternatively, the particle/reaction volume identity can be encoded in the reaction products, e.g., as discussed above for multi-primer nucleic acid amplification methods. Additionally, the two strategies can be combined, encoding groups of separate reaction volumes so that each reaction volume in the group is uniquely identifiable, and then pooling and analyzing each pool individually, as exemplified by the barcoding and pooling methods described above.

颗粒particles

本文描述的方法可用于分析任何类型的颗粒，例如，通过对来自一个或多个单独颗粒的核酸进行任何上述反应。在某些实施方案中，颗粒一般包括足够小以悬浮在流体中、但足够大以从流体区分开的任何物体。颗粒可以是微观的或接近微观的，并可具有约0.005至100μm、0.1至50μm、或约0.5至30μm的直径。可选地或此外，颗粒可具有约10^-20至10^-5克、10^-16至10^-7克、或10^-14至10^-8克的质量。在某些实施方案中，颗粒是来自生物来源的颗粒("生物颗粒")。生物颗粒包括，例如，分子诸如核酸、蛋白、碳水化合物、脂质、和其组合或聚集物(例如，脂蛋白)、以及较大实体，诸如病毒、染色体、细胞囊泡和细胞器、和细胞。如本文所述可分析的颗粒还包括具有不溶组分的那些，例如，在其上附加待分析的分子的珠。Method described herein can be used for analyzing any type of particle, for example, by carrying out any above-mentioned reaction to the nucleic acid from one or more independent particles.In certain embodiments, particle generally comprises enough little to be suspended in fluid but enough large to distinguish any object from fluid.Particle can be microscopic or close to microscopic, and can have the diameter of about 0.005 to 100 μ m, 0.1 to 50 μ m or about 0.5 to 30 μ m.Alternatively or in addition, particle can have the mass of about 10^-20 to 10^-5 gram, 10^-16 to 10^-7 gram or 10^-14 to 10^-8 gram.In certain embodiments, particle is the particle (" bioparticle ") from biological origin.Bioparticle comprises, for example, molecule such as nucleic acid, protein, carbohydrate, lipid and its combination or aggregate (for example, lipoprotein) and larger entity, such as virus, chromosome, cell vesicle and organelle and cell.Particle that can be analyzed as described herein also comprises those with insoluble component, for example, thereon attaches the pearl of molecule to be analyzed.

在示意性实施方案中，颗粒是细胞。适于在本文描述的方法中用作颗粒的细胞一般包括任何自复制、结合膜的生物实体或其任何非复制、结合膜的后代。非复制后代可以是衰老细胞、终末分化细胞、细胞嵌合体、剥夺血清的细胞(serum-starved cell)、感染细胞、非复制突变体、无核细胞、等等。在本文描述的方法中使用的细胞可具有任何来源、遗传背景、健康状态、固定状态、膜透性、预处理、和/或群体纯度以及其他特征。适合的细胞可以是真核、原核、古细菌等等，并可来自动物、植物、真菌、原生生物、细菌、和/或类似来源。在示意性实施方案中，分析人类细胞。细胞可来自生物发育的任何阶段，例如，在哺乳动物细胞(例如，人类细胞)的情形中，可分析胚胎、胎儿或成体细胞。在某些实施方案中，细胞是干细胞。细胞可以是野生型；天然、化学或病毒突变体；改造的突变体(诸如转基因)；和/或类似物。此外，细胞可以是生长的、休眠的、衰老的、转化的、和/或永生的以及其他状态。而且，细胞可以是单种培养(monoculture)，一般从单细胞或非常相似的细胞的小组作为克隆群体获得；可由任何适合的机制预分选，诸如亲和性结合、FACS、药物筛选等等；和/或可以是不同细胞类型的混合或混杂群体。In an illustrative embodiment, the particle is a cell. Cells suitable for use as particles in the methods described herein generally include any self-replicating, membrane-bound biological entity or any non-replicating, membrane-bound offspring thereof. Non-replicating offspring can be senescent cells, terminally differentiated cells, cell chimeras, serum-starved cells, infected cells, non-replicating mutants, anucleated cells, etc. The cells used in the methods described herein can have any source, genetic background, health status, fixed state, membrane permeability, pretreatment, and/or population purity and other characteristics. Suitable cells can be eukaryotic, prokaryotic, archaebacterial, etc., and can come from animals, plants, fungi, protozoa, bacteria, and/or similar sources. In an illustrative embodiment, human cells are analyzed. Cells can be from any stage of biological development, for example, in the case of mammalian cells (e.g., human cells), embryos, fetuses, or adult cells can be analyzed. In certain embodiments, cells are stem cells. Cells can be wild type; natural, chemical, or viral mutants; modified mutants (such as transgenics); and/or analogs. In addition, cells can be growing, dormant, senescent, transformed, and/or immortal, among other states. Furthermore, cells can be a monoculture, typically obtained as a clonal population from a single cell or a group of very similar cells; can be pre-sorted by any suitable mechanism, such as affinity binding, FACS, drug screening, and the like; and/or can be a mixed or heterogeneous population of different cell types.

包括膜(例如，细胞或细胞囊泡或细胞器)、细胞壁或分隔一个或多个内部组分与外部空间的任何其他类型屏障的颗粒可以是完整的或部分(例如，透性化)或完全(例如，以释放内部组分)被破裂的。当颗粒是细胞时，可使用固定的和/或未固定的细胞。活的或死的、固定或未固定的细胞可具有完整的膜、和/或被透性化/破裂的膜，以允许离子、染色剂、染料、标记物、配体等的获取，和/或被溶解以允许细胞内容物的释放。Particles comprising membranes (e.g., cells or cell vesicles or organelles), cell walls, or any other type of barrier separating one or more internal components from the external space may be intact or partially (e.g., permeabilized) or completely (e.g., to release internal components) disrupted. When the particles are cells, fixed and/or unfixed cells may be used. Living or dead, fixed or unfixed cells may have intact membranes, and/or permeabilized/disrupted membranes to allow access to ions, stains, dyes, markers, ligands, etc., and/or be lysed to allow release of cellular contents.

本文描述的方法的一个益处是，它们可用于分析几乎任何数目的颗粒，包括充分低于其他方法所需的数百万颗粒的数目。在多个实施方案中，分析的颗粒数目可以是约10、约50、约100、约500、约1000、约2000、约3000、约4000、约5000、约6000、约7,000、约8000、约9,000、约10,000、约15,000、约20,000、约25,000、约30,000、约35,000、约40,000、约45,000、约50,000、约75,000或约100,000。在具体实施方案中，分析的颗粒数目可落入由以上列出的任何两个数值界定的范围中。One benefit of the methods described herein is that they can be used to analyze virtually any number of particles, including numbers well below the millions required by other methods. In various embodiments, the number of particles analyzed can be about 10, about 50, about 100, about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7,000, about 8000, about 9,000, about 10,000, about 15,000, about 20,000, about 25,000, about 30,000, about 35,000, about 40,000, about 45,000, about 50,000, about 75,000, or about 100,000. In specific embodiments, the number of particles analyzed can fall within a range defined by any two of the values listed above.

颗粒捕获Particle capture

颗粒可通过本领域已知或本文所述的任何手段被捕获在分别的反应体积中。在某些实施方案中，捕获特征保持一个或多个细胞在分别的反应体积中的捕获位点。在优选的实施方案中，捕获特征优先保持仅单细胞在捕获位点。在某些优选的实施方案中，每个捕获位点位于微流体装置的分别的区室中。本文使用的术语"分别的区室"是指至少暂时地与微流体装置中的其他区室分隔的区室，从而该区室可包含分别的反应体积。暂时分隔可例如用阀实现，如从Fluidgm,Inc.(South San Francisco,CA)可得的微流体装置的情形。分隔程度必须使得检验/反应可在区室中分别进行。本文使用的术语"捕获特征"包括串联或平行操作的单个或多个机制(mechanism)。捕获特征可作用以克服由流体流(fluid flow)施加的定位力。适合的捕获特征可以是基于与流偶合的物理屏障(称为"机械捕获")、化学相互作用(称为"基于亲和性的捕获)、真空力、环中的流体流、重力、离心力、磁力、电力(例如，电泳或电渗力)、和/或光学产生的力、及其他。Particles can be captured in separate reaction volumes by any means known in the art or described herein. In certain embodiments, the capture feature maintains a capture site for one or more cells in a separate reaction volume. In a preferred embodiment, the capture feature preferentially maintains only single cells in the capture site. In certain preferred embodiments, each capture site is located in a separate compartment of the microfluidic device. The term "separate compartment" as used herein refers to a compartment that is at least temporarily separated from other compartments in the microfluidic device so that the compartment can contain separate reaction volumes. Temporary separation can be achieved, for example, with a valve, as in the case of the microfluidic device available from Fluidgm, Inc. (South San Francisco, CA). The degree of separation must be such that the test/reaction can be performed separately in the compartment. The term "capture feature" as used herein includes a single or multiple mechanism operating in series or in parallel. The capture feature can act to overcome the positioning force applied by the fluid flow. Suitable capture features can be based on physical barriers coupled to flow (referred to as "mechanical capture"), chemical interactions (referred to as "affinity-based capture"), vacuum forces, fluid flow in a ring, gravity, centrifugal forces, magnetic forces, electrical forces (e.g., electrophoretic or electroosmotic forces), and/or optically generated forces, among others.

捕获特征可以是选择性或非选择性的。选择性机制可以是分级选择性的，即，保持输入的颗粒的少于所有(亚组)。分级选择性机制可至少部分地依赖于随机的集中特征(参见以下)。可选地或此外，选择性机制可以是颗粒依赖性的，即，基于输入的颗粒的一个或多个特性保持颗粒，所述特性诸如尺寸、表面化学、密度、磁性特征、电荷、光学特性(诸如折光率)、和/或类似特性。The capture characteristics can be selective or non-selective. The selective mechanism can be hierarchically selective, i.e., retaining less than all (a subset) of the input particles. The hierarchical selective mechanism can rely, at least in part, on a random concentration characteristic (see below). Alternatively or in addition, the selective mechanism can be particle-dependent, i.e., retaining particles based on one or more characteristics of the input particles, such as size, surface chemistry, density, magnetic characteristics, charge, optical properties (such as refractive index), and/or the like.

机械捕获Mechanical capture

机械捕获可至少部分地基于颗粒与例如微流体装置中布置的任何适合的物理屏障接触。这种颗粒-屏障接触一般限制沿着流体流方向的纵向颗粒移动，产生流动辅助的保持(flow-assisted retention)。流动辅助的颗粒-屏障接触还可限制侧对侧的/正交(横向)移动。适合的物理屏障可由从通道或其他通路的任何部分(即，壁、顶和/或底)向内延伸的凸出物形成。例如，凸出物可以是固定的和/或可移动的，包括柱(column)、杆(post)、块(block)、凸块(bump)、壁、和/或部分/完全封闭的阀、及其他。一些物理屏障诸如阀，可以是可移动或可调整的。可选地或此外，物理屏障可由在通道或其他通路中形成的凹处(例如，龛)、或由流体可渗透膜界定。其他物理屏障可基于通路的横截面尺寸形成。例如，尺寸选择性通道可保持过大不能进入通道的颗粒。(尺寸选择性通道还可称为过滤通道、微通道、或颗粒限制性或颗粒选择性通道。)实施例6和8提供示例性的机械捕获实施方案。Mechanical capture can be based, at least in part, on contact of particles with any suitable physical barrier disposed, for example, in a microfluidic device. Such particle-barrier contact generally restricts longitudinal particle movement along the direction of fluid flow, resulting in flow-assisted retention. Flow-assisted particle-barrier contact can also restrict side-to-side/orthogonal (lateral) movement. Suitable physical barriers can be formed by projections extending inwardly from any portion of a channel or other passageway (i.e., a wall, top, and/or bottom). For example, projections can be fixed and/or movable, including columns, posts, blocks, bumps, walls, and/or partially/fully enclosed valves, among others. Some physical barriers, such as valves, can be movable or adjustable. Alternatively or in addition, a physical barrier can be formed by a recess (e.g., a niche) formed in a channel or other passageway, or defined by a fluid-permeable membrane. Other physical barriers can be formed based on the cross-sectional dimensions of the passageway. For example, a size-selective passageway can retain particles that are too large to enter the passageway. (Size-selective channels may also be referred to as filtration channels, microchannels, or particle-restricting or particle-selective channels.) Examples 6 and 8 provide exemplary mechanical capture embodiments.

基于亲和性的捕获Affinity-based capture

基于亲和性的捕获可基于一种或多种化学相互作用保持颗粒，即，其中结合伴侣结合颗粒组分。化学相互作用可以是共价和/或非共价的相互作用，包括离子、静电、疏水、范德华、和/或金属配位相互作用、及其他。化学相互作用可选择性和/或非选择性地保持颗粒。选择性和非选择性保持可基于颗粒和表面之间的特异性和/或非特异性化学相互作用，例如，在微流体装置中。Affinity-based capture can retain particles based on one or more chemical interactions, i.e., where a binding partner binds to a particle component. Chemical interactions can be covalent and/or non-covalent interactions, including ionic, electrostatic, hydrophobic, van der Waals, and/or metal coordination interactions, among others. Chemical interactions can selectively and/or non-selectively retain particles. Selective and non-selective retention can be based on specific and/or non-specific chemical interactions between the particle and the surface, for example, in a microfluidic device.

特异性化学机制可使用特异性结合伴侣(SBP)，例如，颗粒和装置表面上分别布置的第一和第二SBP。示例性SBP可包括生物素/抗生物素蛋白、抗体/抗原、凝集素/碳水化合物等。SBP可在装置形成之前、期间和/或之后局部地布置在微流体装置中。例如，基质和/或流体层组分的表面可在基质和流体层组分被连接之前被SBP成员的粘附/附加来局部修饰。可选地或此外，在已经形成装置后SBP可局部地与微流体装置的部分缔合，例如，通过SBP成员与装置的局部化学反应(诸如用光的局部光照催化的化学反应)。还参见实施例7，其描述一个实施方案，其中带有SBP成员的珠被机械捕捉在捕获位点以展示SBP成员用于基于亲和性的捕获颗粒(即，细胞)。Specific chemical mechanisms can use specific binding partners (SBPs), for example, first and second SBPs disposed on the surface of a particle and a device, respectively. Exemplary SBPs can include biotin/avidin, antibodies/antigens, lectins/carbohydrates, etc. The SBPs can be locally disposed in a microfluidic device before, during, and/or after device formation. For example, the surfaces of a matrix and/or fluid layer component can be locally modified by adhesion/attachment of SBP members before the matrix and fluid layer components are connected. Alternatively or in addition, the SBPs can be locally associated with portions of a microfluidic device after the device has been formed, for example, by local chemical reactions of the SBP members with the device (such as chemical reactions catalyzed by local illumination with light). See also Example 7, which describes an embodiment in which beads bearing SBP members are mechanically captured at capture sites to display the SBP members for affinity-based capture of particles (i.e., cells).

非特异性化学机制可依赖于微流体装置的表面化学的局部差异。如上所述，此类局部差异可在微流体装置形成之前、期间和/或之后产生。局部差异可来自局部的化学反应，例如，以产生疏水或亲水区、和/或材料的局部结合。结合的材料可包括聚-L-赖氨酸、聚-D-赖氨酸、聚乙烯亚胺、白蛋白、明胶、胶原、层粘连蛋白、纤连蛋白、巢蛋白(entactin)、玻璃粘连蛋白(vitronectin)、原纤维蛋白、弹性蛋白、肝素、硫酸角质素、硫酸乙酰肝素、硫酸软骨素、透明质酸、和/或细胞外基质提取物/混合物、及其他。Non-specific chemical mechanisms can rely on local differences in the surface chemistry of the microfluidic device. As mentioned above, such local differences can occur before, during, and/or after the microfluidic device is formed. Local differences can come from local chemical reactions, for example, to produce hydrophobic or hydrophilic regions, and/or local binding of materials. The combined materials can include poly-L-lysine, poly-D-lysine, polyethyleneimine, albumin, gelatin, collagen, laminin, fibronectin, entactin, vitronectin, fibrillin, elastin, heparin, keratan sulfate, heparan sulfate, chondroitin sulfate, hyaluronic acid, and/or extracellular matrix extracts/mixtures and other.

其他捕获特征Other capture features

可选地或除了基于亲和性的或机械捕获以外，可使用其他捕获特征。这些机制的一些或所有、和/或上述机制可至少部分地依赖于颗粒与微流体装置通道或通路之间的摩擦来帮助保持。Alternatively or in addition to affinity-based or mechanical capture, other capture features may be used. Some or all of these mechanisms, and/or the mechanisms described above, may rely at least in part on friction between the particle and a microfluidic device channel or passageway to aid retention.

捕获特征可基于真空力、流体流和/或重力。基于真空的捕获特征可施加牵拉颗粒与通路表面更紧密接触的力，例如利用从通道向外的力。真空施加、和/或颗粒保持，可由通道或其他通路壁中的孔/口来帮助。相反，基于流体流的捕获特征可产生保持颗粒的流体流路径诸如环。这些流体流路径可由以下形成：无出口的闭合通道回路(例如，由阀关闭和主动泵送)，和/或涡流，诸如由凹处中一般环状流体流形成的。基于重力的捕获特征可对通路的底表面保持颗粒，从而与摩擦组合来限制颗粒移动。基于重力的保持可由凹处和/或减少流体流速来帮助。The capture feature can be based on vacuum force, fluid flow and/or gravity. The vacuum-based capture feature can apply a force that pulls the particles into closer contact with the passage surface, for example, by utilizing a force outward from the passage. Vacuum application, and/or particle retention, can be assisted by holes/ports in the passage or other passage walls. In contrast, the capture feature based on fluid flow can produce fluid flow paths such as rings that retain particles. These fluid flow paths can be formed by: closed passage loops with no outlet (for example, by valve closure and active pumping), and/or eddies, such as those formed by a generally annular fluid flow in a recess. The gravity-based capture feature can retain particles on the bottom surface of the passage, thereby limiting particle movement in combination with friction. Gravity-based retention can be assisted by recesses and/or reducing fluid flow rates.

捕获特征可基于离心力、磁力、和/或光学产生的力。基于离心力的捕获特征可通过对通路表面推颗粒来保持颗粒，通常通过对颗粒施加一般垂直于流体流的力。这样的力可通过离心微流体装置和/或通过在流体流路径中的颗粒移动来施加。基于磁力的捕获特征可利用在微流体装置内部和/或外部产生的磁场保持颗粒。磁场可与颗粒的铁磁性和/或顺磁性部分相互作用。例如，珠可至少部分地由铁磁性材料形成，或细胞可包括表面结合的或内化的铁磁性颗粒。基于电力的捕获特征可利用电场保持带电荷的颗粒和/或群体。相反，基于光学产生的力操作的捕获特征可利用光来保持颗粒。此类机制可基于光镊及其他的原理操作。Capture features can be based on centrifugal, magnetic, and/or optically generated forces. Centrifugal-based capture features can hold particles by pushing them against the surface of the pathway, typically by applying a force generally perpendicular to the fluid flow to the particles. Such forces can be applied by centrifugal microfluidic devices and/or by particle movement in the fluid flow path. Magnetic-based capture features can utilize magnetic fields generated inside and/or outside the microfluidic device to hold particles. The magnetic field can interact with the ferromagnetic and/or paramagnetic parts of the particles. For example, beads can be formed at least in part of a ferromagnetic material, or cells can include surface-bound or internalized ferromagnetic particles. Electric-based capture features can utilize electric fields to hold charged particles and/or colonies. In contrast, capture features based on optically generated force operations can utilize light to hold particles. Such mechanisms can operate based on the principles of optical tweezers and others.

捕获特征的另一种形式是盲填充(blind-fill)通道，其中通道具有入口、但不变地(fixedly)或暂时地没有出口。例如，当微流体装置由气透性材料诸如PDMS制造时，当被经入口的液体的入流迫使离开时，死端通道中存在的气体可逃逸，或被迫使经气透性材料离开通道。这是盲填充的优选的实例。盲填充可与具有入口、和被门控或装有阀(valved bya valve)的出口的通道或区室一起使用。在这一实例中，当关闭出口阀同时经入口填充通道或区室时，发生填充气体的通道或区室的盲填充。如果入口也具有阀，在完成盲填充后可随后关闭该阀，然后可打开出口以将通道或区室内容物暴露于另一通道或区室。如果第三入口与通道或区室连通，该第三入口可将另外的流体、气体或液体引入通道或区室来以测量的量排出待从通道或区室排出的盲填充液体。Another form of capture feature is blind filling (blind-fill) channel, wherein channel has inlet but does not have outlet fixedly or temporarily.For example, when microfluidic device is made of gas permeable material such as PDMS, when being forced to leave by the inflow of the liquid through the inlet, the gas present in the dead end channel can escape, or is forced to leave the channel through the gas permeable material.This is the preferred example of blind filling.Blind filling can be used with the channel or the chamber with inlet and the outlet that is gated or valve (valved by a valve) is housed.In this example, when closing the outlet valve while filling channel or chamber through the inlet, the blind filling of the channel or chamber of gas filling occurs.If the inlet also has a valve, the valve can be closed subsequently after completing blind filling, then the outlet can be opened to expose channel or chamber content to another channel or chamber.If the 3rd inlet is communicated with channel or chamber, the 3rd inlet can introduce other fluid, gas or liquid into channel or chamber to discharge the blind filling liquid to be discharged from channel or chamber with measured amount.

集中特征(Focusing Feature)Focusing Feature

微流体装置中通过使用一个或多个集中特征以集中颗粒流到每个捕获位点可增强颗粒捕获。集中特征可非限制性地以多种方式分类，例如，以反映其来源和/或操作原理，包括直接和/或间接、流体介导的和/或非流体介导的、外部和/或内部的、等等。这些分类不是互相排除的。如此，给定的集中特征可以两种或更多种方式定位颗粒；例如，电场可直接(例如，经由电泳)和间接(例如，经由电渗)定位颗粒。Particle capture can be enhanced in a microfluidic device by using one or more focusing features to focus the flow of particles to each capture site. Focusing features can be classified in a variety of ways, for example, to reflect their source and/or operating principle, including direct and/or indirect, fluid-mediated and/or non-fluid-mediated, external and/or internal, etc., without limitation. These classifications are not mutually exclusive. Thus, a given focusing feature can position particles in two or more ways; for example, an electric field can position particles directly (e.g., via electrophoresis) and indirectly (e.g., via electroosmosis).

集中特征可作用以纵向地和/或横向地界定颗粒位置。术语"纵向位置"表示平行于或沿着微流体通道的长轴和/或通道中的流体流动流(fluid flow stream)的位置。相反，术语"横向位置"表示垂直于通道的长轴和/或相关的主要流体流动流的位置。通过在弯曲通道中使"长轴"与"切线"相等，纵向和横向位置二者可被局部地界定。集中特征可作用以在纵向和横向流之间、沿着相对于通道的长轴和/或流体流以任何角度的路径移动颗粒。The concentrated feature can act to define the particle position longitudinally and/or laterally. The term "longitudinal position" refers to a position parallel to or along the long axis of the microfluidic channel and/or the fluid flow stream in the channel. In contrast, the term "lateral position" refers to a position perpendicular to the long axis of the channel and/or the associated main fluid flow stream. By equating the "long axis" with the "tangent" in a curved channel, both the longitudinal and lateral positions can be locally defined. The concentrated feature can act to move particles between the longitudinal and lateral flows, along a path at any angle relative to the long axis of the channel and/or the fluid flow.

集中特征可单独和/或组合使用。如果组合使用，各特征可连续地(即，顺序地)和/或平行地(即，同时)使用。例如，间接机制诸如流体流可用于粗略定位，直接机制诸如光镊可用于最终定位。The focusing features can be used individually and/or in combination. If used in combination, the features can be used sequentially (i.e., sequentially) and/or in parallel (i.e., simultaneously). For example, an indirect mechanism such as fluid flow can be used for coarse positioning, and a direct mechanism such as optical tweezers can be used for final positioning.

直接集中特征一般包括其中力直接作用于颗粒以将颗粒定位在微流体网络中的任何机制。直接集中特征可基于任何适合的机制，包括基于光、电、磁和/或重力的力，及其他。光学集中特征利用光来介导或至少帮助定位颗粒。适合的光学集中特征包括"光镊"，其利用适当集中的和可移动的光源来对颗粒给予定位力。电集中特征利用电来定位颗粒。适合的电机制包括"动电学"，即，跨微流体网络的一些或全部施加电压和/或电流，如上所述的，其可直接(例如，经由电泳)和/或间接地经由流体中离子的移动(例如，经由电渗)移动带电荷的颗粒。磁性集中特征使用磁性以基于磁性相互作用定位颗粒。适合的磁性机制包括在流体网络中或周围施加磁场，以经由其与颗粒中、颗粒上或颗粒附近的铁磁性和/或顺磁性材料的缔合定位颗粒。基于重力的集中特征利用重力来定位颗粒，例如，将附着细胞与在细胞培养物位置的基质接触。Direct concentration features generally include any mechanism in which a force acts directly on a particle to position the particle in a microfluidic network. Direct concentration features can be based on any suitable mechanism, including forces based on light, electricity, magnetism and/or gravity, and others. Optical concentration features use light to mediate or at least help position particles. Suitable optical concentration features include "optical tweezers," which use a properly concentrated and movable light source to impart a positioning force to particles. Electrical concentration features use electricity to position particles. Suitable electrical mechanisms include "electrokinetics," i.e., applying a voltage and/or current across some or all of the microfluidic network, as described above, which can move charged particles directly (e.g., via electrophoresis) and/or indirectly via the movement of ions in the fluid (e.g., via electroosmosis). Magnetic concentration features use magnetism to position particles based on magnetic interactions. Suitable magnetic mechanisms include applying a magnetic field in or around the fluid network to position particles via its association with ferromagnetic and/or paramagnetic materials in, on, or near the particles. Gravity-based focusing features utilize gravity to position particles, for example, to place attached cells in contact with a matrix in a cell culture location.

间接集中特征一般包括其中力间接作用于颗粒的任何机制，例如，经由流体纵向地和/或横向地移动微流体网络中的颗粒。纵向间接集中特征一般可由沿着通道和/或其他通路的流体流产生和/或调整。因此，纵向集中特征可被调整流速和/或路径的阀和/或泵帮助和/或调整。在一些情形中，纵向集中特征可被电渗集中特征帮助和/或调整。可选地或此外，纵向集中特征可以是基于输入的，即，被输入机制帮助和/或调整，所述输入机制诸如基于压力或重力的机制，包括由流体柱的不等高度产生的压头(pressure head)。Indirect concentration characteristics generally include any mechanism in which force acts indirectly on particles, for example, longitudinally and/or laterally moving particles in the microfluidic network via fluid. The longitudinal indirect concentration characteristics generally can be produced and/or adjusted by the fluid flow along the channel and/or other paths. Therefore, the longitudinal concentration characteristics can be helped and/or adjusted by valves and/or pumps that adjust flow rate and/or path. In some cases, the longitudinal concentration characteristics can be helped and/or adjusted by electroosmotic concentration characteristics. Alternatively or in addition, the longitudinal concentration characteristics can be input-based, that is, helped and/or adjusted by an input mechanism, such as a mechanism based on pressure or gravity, including a pressure head (pressure head) produced by the unequal heights of the fluid column.

横向间接集中特征一般可被在通道接头、侧面布置的减少的流体流区、通道弯曲、和/或物理屏障(即，阻碍物)处的流体流动流产生和/或调整。基于携带朝向位点的流体的通道数目相对于携带离开位点的流体的通道数目，通道接头可以是统一的位点(unifyingsites)或分隔的位点(dividing sites)。物理屏障可具有任何适合的设计以引导颗粒朝向捕获位点流动。例如，阻碍物可从任何通道表面向外延伸，例如，以引导颗粒朝向捕获位点流动的角度。可调整阻碍物长度、与通道表面的角度、和与捕获位点的距离以增强颗粒朝向捕获位点的流动。阻碍物可由从通道或其他通路的任何部分(即，壁、顶和/或底)向内延伸的凸出物形成。例如，凸出物可以是固定的和/或可移动的，包括柱、杆、块、凸块、壁、和/或部分/完全封闭的阀、及其他。一些物理屏障诸如阀，可以是可移动或可调整的。The lateral indirect concentration feature can generally be produced and/or adjusted by the fluid flow stream at the channel joint, the reduced fluid flow area arranged on the side, the channel bend, and/or the physical barrier (i.e., obstruction). Based on the number of channels carrying the fluid towards the site relative to the number of channels carrying the fluid leaving the site, the channel joint can be a unified site (unifying sites) or a separated site (dividing sites). The physical barrier can have any suitable design to guide the particles to flow towards the capture site. For example, the obstruction can extend outward from any channel surface, for example, to guide the particles to flow towards the capture site. The obstruction length, the angle with the channel surface and the distance with the capture site can be adjusted to enhance the flow of particles towards the capture site. The obstruction can be formed by a projection extending inward from any part (i.e., wall, top and/or bottom) of the channel or other passages. For example, the projection can be fixed and/or movable, including a column, a rod, a block, a projection, a wall, and/or a partially/completely enclosed valve and other. Some physical barriers, such as valves, can be movable or adjustable.

在一些实施方案中，对每个捕获位点可采用多个阻碍物。例如，可采用从通道的每个侧壁以一定角度向外延伸的阻碍物以引导颗粒朝向在通道中心定位的捕获位点流动。参见图22A-B。当采用机械捕获时，阻碍物可以与捕获位点中的物理屏障间隔。可选地或另外，阻碍物可接触捕获位点中的物理屏障或是捕获位点中的物理屏障的固有部分。参见图22A和14C。例如，以一定角度从通道壁向外延伸的阻碍物可接触凹的捕获特征(例如，物理屏障)或是凹的捕获特征(例如，物理屏障)的固有部分。应理解的是，"凹的"捕获特征在捕获特征的一般面对流体流方向的一侧是凹的。阻碍物导引颗粒离开通道壁和朝向凹的捕获特征流动，帮助颗粒捕获。沿着流路径的下一个捕获位点可具有相似的阻碍物-凹的捕获特征构造，阻碍物从通道的相同的壁延伸。然而，在一些实施方案中，下一个阻碍物-凹的捕获特征从相对的通道壁延伸是有利的。这一交替的构造作用以集中从一个阻碍物到下一个阻碍物的流，从而沿着每个阻碍物的流增强进入每个凹的捕获特征的颗粒流。参见图22C。In some embodiments, multiple obstructions may be used for each capture site. For example, an obstruction extending outward at an angle from each side wall of the channel may be used to guide the flow of particles toward a capture site located in the center of the channel. See Figures 22A-B. When mechanical capture is used, the obstruction may be spaced apart from the physical barrier in the capture site. Alternatively or in addition, the obstruction may contact the physical barrier in the capture site or be an inherent part of the physical barrier in the capture site. See Figures 22A and 14C. For example, an obstruction extending outward at an angle from the channel wall may contact a concave capture feature (e.g., a physical barrier) or be an inherent part of a concave capture feature (e.g., a physical barrier). It should be understood that a "concave" capture feature is concave on the side of the capture feature that generally faces the direction of fluid flow. The obstruction guides the particles away from the channel wall and toward the concave capture feature, assisting in particle capture. The next capture site along the flow path may have a similar obstruction-concave capture feature configuration, with the obstruction extending from the same wall of the channel. However, in some embodiments, it is advantageous for the next obstruction-concave capture feature to extend from the opposite channel wall. This alternating configuration acts to focus the flow from one obstruction to the next, thereby enhancing the flow of particles into each concave capture feature along the flow of each obstruction. See Figure 22C.

横向间接集中特征可基于层流、随机分隔、和/或离心力以及其他机制。微流体装置中颗粒和/或试剂的横向定位可至少部分地被基于层流的机制介导。基于层流的机制一般包括其中通道中输入流动流的位置由通道中另外的流动流的存在、不存在和/或相对位置来确定的任何集中特征。这种基于层流的机制可由为统一位点的通道接头界定，在统一位点处来自两个、三个或更多个通道的入口流动流流向接头，统一以形成流动离开接头的较少量出口流动流，优选地1个。由于在微流体尺度的流动流的层流特性，统一位点在入口流动流统一为分层出口流动流后可保持入口流动流的相对分布。因此，颗粒和/或试剂可保持局限于任何所选的一个或多个层流流，入口通道基于其携带颗粒和/或试剂，从而横向地定位颗粒和/或试剂。参见，例如，图24D。Lateral indirect concentration feature can be based on laminar flow, random separation and/or centrifugal force and other mechanisms.The lateral positioning of particle and/or reagent in microfluidic device can be mediated at least in part by the mechanism based on laminar flow.The mechanism based on laminar flow generally comprises any concentrated feature that the position of wherein input flow stream in channel is determined by the existence, non-existence and/or relative position of other flow stream in channel.This mechanism based on laminar flow can be defined by the channel joint for unified site, at unified site, the inlet flow stream from two, three or more channels flows to joint, unifies to form a less amount of outlet flow stream, preferably 1, that flows away from joint.Due to the laminar flow characteristic of the flow stream at microfluid scale, unified site can keep the relative distribution of inlet flow stream after inlet flow stream is unified into layered outlet flow stream.Therefore, particle and/or reagent can keep being confined to any selected one or more laminar flows, and inlet channel carries particle and/or reagent based on it, thereby positions particle and/or reagent laterally.Referring to, for example, Figure 24 D.

每个入口流动流的相对尺寸(或流速)和位置可决定携带颗粒和/或试剂的流动流的位置和相对宽度二者。例如，相对小的(窄的)、侧面为两个较大(较宽)流动流的颗粒/试剂的入口流动流，可占据单个出口通道中窄的中央位置。相反，相对大的(宽的)、侧面为尺寸相当的流动流和较小(较窄)流动流的颗粒/试剂的入口流动流，可占据横向地朝较小流动流偏置的较宽的位置。在任一种情形中，基于层流的机制可称为集中机制，因为颗粒/试剂被"集中"到出口通道的横截面积的亚组。可使用基于层流的机制以单独地运送(address)颗粒和/或试剂到多个不同的捕获位点。In some embodiments, the inlet flow stream of the present invention can be used to carry out the flow of the particle/reagent of the present invention.The relative size (or flow velocity) and the position of each inlet flow stream can determine the position and relative width of the flow stream carrying particle and/or reagent.For example, relatively small (narrow), the side is the inlet flow stream of the particle/reagent of two larger (wider) flow streams, can occupy narrow central position in single outlet channel.On the contrary, relatively large (wide), the side is the inlet flow stream of the particle/reagent of the flow stream of suitable size and less (narrower) flow stream, can occupy the wider position of laterally biasing towards less flow stream.In any case, the mechanism based on laminar flow can be called a centralized mechanism, because particle/reagent is " concentrated " to the subgroup of the cross-sectional area of outlet channel.Can use the mechanism based on laminar flow to transport (address) particle and/or reagent to a plurality of different capture sites individually.

基于层流的机制可以是可变的机制以改变颗粒/试剂的横向位置。如上所述，每个入口流动流的相对贡献可决定颗粒/试剂流动流的横向位置。任何入口流动流的改变的流可改变其对出口流动流的贡献，相应转变颗粒/试剂流动流。在称为灌注机制(perfusionmechanism)的极端的情形中，可横向地移动试剂(或颗粒)流动流，基于存在或不存在来自邻近的入口流动流的流，与保持的颗粒(试剂)接触或与保持的颗粒(试剂)分开。这样的机制还可用于实现颗粒的可变的或调整的横向定位，例如，以引导颗粒向具有不同的横向位置的捕获位点。The mechanism based on laminar flow can be a variable mechanism to change the lateral position of particle/reagent. As mentioned above, the relative contribution of each inlet flow stream can determine the lateral position of particle/reagent flow stream. The stream of the change of any inlet flow stream can change its contribution to outlet flow stream, corresponding transformation particle/reagent flow stream. In the extreme situation called perfusion mechanism (perfusion mechanism), reagent (or particle) flow stream can be moved laterally, based on the presence or absence of stream from adjacent inlet flow stream, contact with the particle (reagent) maintained or separate with the particle (reagent) maintained. Such mechanism can also be used to realize the variable or lateral positioning of particle, for example, to guide particle to capture sites with different lateral positions.

微流体装置中颗粒和/或试剂的横向定位可至少部分地由随机(或分部流(portioned flow))集中特征介导。随机横向集中特征一般包括其中至少部分地随机选择的输入的颗粒或试剂的亚组被侧向分配离开主要流动流到通道中减少的流体流的区域(或可能地，到不同的通道)的任何集中特征。减少的流的区域可促进颗粒保持、处理、检测、将颗粒损伤最小化、和/或促进颗粒与基质接触。随机集中特征可由分隔流位点和/或局部加宽以及其他的通道决定。The lateral positioning of particles and/or reagents in microfluidic devices can be mediated at least in part by random (or portioned flow) concentrated features. Random lateral concentrated features generally include any concentrated features in which a subset of particles or reagents of an input selected at least in part at random is laterally distributed to leave the main flow stream to a region (or possibly, to different channels) of reduced fluid flow in the channel. The region of reduced flow can promote particle retention, processing, detection, minimize particle damage, and/or promote particle to contact with matrix. Random concentrated features can be determined by separating flow sites and/or local widening and other channels.

分隔流位点可通过形成减少的流体流速的区域来实现随机定位。分隔流位点一般包括任何通道接头，在此处来自一个(优选地)或多个入口通道的入口流动流被分隔到较大数目的出口通道中，包括两个、三个或更多个通道。这样的分隔位点可传递颗粒的亚组到在接头处或附近形成的减少的流速或准-停滞流的区域，所述颗粒的亚组可随机选择或基于颗粒的特性(诸如质量)选择。由亚组代表的颗粒的比例可依赖于出口通道相对于入口通道的相对流方向。这些流方向可一般垂直于入口流动流，以相对方向被导向，以形成"T-接头"。可选地，出口流方向可形成小于和/或大于90度的角度。The separation flow site can realize random positioning by forming the region of the fluid flow rate that reduces.The separation flow site generally comprises any channel joint, and the inlet flow stream from one (preferably) or multiple inlet channels is separated into the outlet channel of larger number, comprises two, three or more channels here.Such separation site can transmit the subset of particle to the flow velocity of the reduction formed at or near the joint or the region of quasi-stagnant flow, and the subset of described particle can be randomly selected or selected based on the characteristic (such as quality) of particle.The ratio of the particle represented by the subset can depend on the relative flow direction of outlet channel with respect to inlet channel.These flow directions can be generally perpendicular to the inlet flow stream, are guided in relative direction, to form " T-joint ".Alternatively, the outlet flow direction can form an angle less than and/or greater than 90 degree.

具有两个或更多个出口通道的分隔流集中特征可用作分部流机制。具体地，被携带到通道接头的流体、颗粒和/或试剂可按照经两个或更多个出口通道的流体流被分部。因此，进入两个或更多个通道的颗粒或试剂的数目或体积比例可被通道的相对尺寸和/或经通道的流体流速调整，通道的相对尺寸和/或经通道的流体流速转而可被阀或其他适合的流调整机制调整。在第一组实施方案中，出口通道可以是非常不等的尺寸，从而仅有小比例的颗粒和/或试剂被导向较小的通道。在第二组实施方案中，阀可用于形成试剂的期望稀释。在第三组实施方案中，阀可用于选择性导向颗粒到两个或更多个流体路径之一。The separation flow with two or more outlet channels concentrates feature and can be used as the branch flow mechanism.Particularly, the fluid, particle and/or reagent that are carried to the channel joint can be divided according to the fluid flow through two or more outlet channels.Therefore, the particle that enters two or more channels or number or the volume ratio of reagent can be adjusted by the relative size of channel and/or the fluid flow rate through channel, and the relative size of channel and/or the fluid flow rate through channel can in turn be adjusted by valve or other applicable stream adjustment mechanisms.In the first group of embodiments, outlet channels can be very unequal sizes, thereby only have small proportion of particle and/or reagent to be directed to less channel.In the second group of embodiments, valve can be used for forming the expectation dilution of reagent.In the third group of embodiments, valve can be used for selectivity guide particle to one of two or more fluid paths.

局部加宽的通道可通过产生主要流动流侧向的降低流速的区域促进随机定位。降低流速可在降低流速的区域沉降输入的颗粒的亚组。这样的加宽的通道可包括以一定角度弯曲(curve)或弯下(bend)的非直线通道。可选地或此外，加宽的区域可由在通道壁、横贯通道的室和/或等等中形成的凹处形成，尤其在弯曲或弯下的通道的外侧缘。The passage of local widening can promote random positioning by the region of the reduction flow velocity that produces the main flow stream lateral.Reducing the flow velocity can be in the subgroup of the particle of the regional sedimentation input of reducing the flow velocity.The passage of such widening can comprise the non-straight channel that bends (curve) or bends (bend) with a certain angle.Alternatively or in addition, the zone of widening can be formed by the recess that forms in the chamber of channel wall, traverse passage and/or etc., especially at the outer side edge of the passage that bends or bends.

颗粒和/或试剂的横向定位还可至少部分地被离心集中特征介导。在离心集中特征中，颗粒可经历由速度改变决定的离心力，例如，通过移动经过流体路径中的弯道。颗粒的尺寸和/或密度可决定速度改变的速率，分配不同尺寸和/或密度的颗粒到不同横向位置。The lateral positioning of particles and/or reagents can also be mediated, at least in part, by a centrifugal focusing feature. In a centrifugal focusing feature, particles can experience centrifugal forces determined by changes in velocity, for example, by moving through bends in the fluid path. The size and/or density of the particles can determine the rate of change in velocity, distributing particles of different sizes and/or densities to different lateral positions.

引流沟特征Drainage ditch characteristics

在某些实施方案中，捕获位点还包括引流沟特征。当采用机械捕获时，例如，引流沟特征可包括捕获特征中的一个或多个障碍物，其尺寸为允许流体流动但不允许颗粒流动经过和/或围绕捕获特征。如此，例如，捕获特征可包括由空间(引流沟特征)间隔的两个物理屏障，其中空间足够大以允许无颗粒的流体以足够低的阻抗在屏障之间流动以引导细胞朝向屏障，从而增强颗粒捕获的概率。物理屏障之间的空间一般应足够小和/或适当地构造，使得将在捕获位点被捕获的颗粒将不穿过屏障。在具体、示例性实施方案中，捕获特征包括带有第一和第二端的两个凹的物理屏障，其中屏障布置为在屏障的第一端之间带有小空间，形成引流沟特征，在屏障的第二端之间带有较大空间。参见图22B(其中d3大于d1，d1形成引流沟)。以这一构造，屏障形成尺寸适合捕获颗粒的"杯"，在杯的基部带有引流沟。凭借引流沟，颗粒朝向杯流动，只要其未被占据。颗粒流入杯中后，引流沟被"塞上"，这倾向于增强颗粒围绕杯的流动并继续到微流体装置中的下一捕获特征。In certain embodiments, the capture site also includes a drainage groove feature. When mechanical capture is employed, for example, the drainage groove feature may include one or more obstacles in the capture feature that are sized to allow fluid flow but not particles to flow through and/or around the capture feature. Thus, for example, the capture feature may include two physical barriers separated by a space (the drainage groove feature), where the space is large enough to allow particle-free fluid to flow between the barriers with sufficiently low impedance to guide cells toward the barriers, thereby increasing the probability of particle capture. The space between the physical barriers should generally be small enough and/or appropriately configured so that particles that would be captured at the capture site will not pass through the barriers. In a specific, exemplary embodiment, the capture feature includes two concave physical barriers with first and second ends, where the barriers are arranged with a small space between the first ends of the barriers, forming the drainage groove feature, and a larger space between the second ends of the barriers. See Figure 22B (where d3 is greater than d1, d1 forming the drainage groove). With this configuration, the barriers form a "cup" sized to capture particles, with a drainage groove at the base of the cup. Particles flow toward the cup due to the drainage groove, as long as it is unoccupied. After the particles flow into the cup, the drainage channels are "plugged," which tends to enhance the flow of particles around the cup and on to the next capture feature in the microfluidic device.

未优化的单颗粒捕获Unoptimized single particle capture

在具体实施方案中，捕获技术诸如有限稀释用于在分别的反应体积中捕获颗粒。在这一类型的捕获中，例如在微流体装置中没有使用任何捕获特征，诸如优先保持仅单细胞在捕获位点的结合亲和性或机械特征。例如，有限稀释可如下进行：通过制备一系列颗粒悬液的稀释液，并将来自每个稀释液的小份分配到分别的反应体积中。确定每个反应体积中颗粒的数目，然后选择产生具有仅单颗粒的反应体积的最高比例的稀释液并将其用于对本文描述的参数测量捕获颗粒。In a specific embodiment, a capture technique such as limiting dilution is used to capture particles in separate reaction volumes. In this type of capture, no capture characteristics, such as binding affinity or mechanical characteristics that preferentially retain only single cells at the capture site, are used, for example, in a microfluidic device. For example, limiting dilution can be performed as follows: by preparing a series of dilutions of a particle suspension and distributing a small aliquot from each dilution into separate reaction volumes. The number of particles in each reaction volume is determined, and then the dilution that produces the highest proportion of reaction volumes with only single particles is selected and used to capture particles for the parameter measurements described herein.

优化的单颗粒捕获Optimized single particle capture

在一些实施方案中，方法包括使用优化的捕获技术来增加高于利用诸如有限稀释的方法实现的具有仅一个颗粒的分别的反应体积的预计比例(即，高于约33％)。在这些实施方案的变化形式中，捕获被优化使得各自具有仅一个颗粒的分别的反应体积的预计比例是分别的反应体积的总数目的至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％或至少约95％。在具体实施方案中，各自具有仅一个颗粒的分别的反应体积的预计比例落入以以上列出的任何两个百分比为边界的范围中。各自具有仅一个颗粒的分别的反应体积的预计比例可通过经验或统计学手段确定，取决于特定捕获技术(例如，有限稀释以与泊松分布一致的方式产生具有仅一个颗粒的反应体积)。本文使用的术语"优化"不暗示实现最优的结果，仅表示采取一些措施来增加具有仅一个颗粒的分别的反应体积的预计比例高于约33％。在具体实施方案中，例如，利用排除在每个反应体积(捕获位点)中多于一个颗粒的保持的基于尺寸的机制，可实现优化的单颗粒捕获。In some embodiments, the method includes using an optimized capture technology to increase the expected proportion of each reaction volume with only one particle (i.e., higher than about 33%) achieved using methods such as limiting dilution. In variations of these embodiments, capture is optimized so that the expected proportion of each reaction volume with only one particle is at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% of the total number of reaction volumes. In specific embodiments, the expected proportion of each reaction volume with only one particle falls within a range bounded by any two percentages listed above. The expected proportion of each reaction volume with only one particle can be determined empirically or statistically, depending on the specific capture technology (e.g., limiting dilution produces reaction volumes with only one particle in a manner consistent with a Poisson distribution). As used herein, the term "optimize" does not imply achieving optimal results, but merely means taking some steps to increase the expected proportion of respective reaction volumes with only one particle above about 33%. In specific embodiments, optimized single particle capture can be achieved, for example, using a size-based mechanism that precludes the retention of more than one particle per reaction volume (capture site).

在某些实施方案中，机械捕获单独使用或与一个或多个其他捕获特征组合使用以在每个分别的反应体积(即，微流体装置中的每个捕获位点)中优先捕获单颗粒。例如，每个捕获位点可包括尺寸为包含仅一个颗粒的一个或多个物理屏障。物理屏障的形状可设计为增强颗粒的保持。例如，当颗粒是细胞时，物理屏障的尺寸和构造可形成适于保持仅一个细胞的凹的表面。在此类实施方案中，可设计物理屏障从而允许流体流经未被细胞占据时的捕获位点，和/或捕获位点可包括帮助这一流动的引流沟特征。在具体实施方案中，微流体装置包含多个适当地尺寸/构造的物理屏障，藉以多个单独颗粒被保持在装置中，每个物理屏障保持一个颗粒。在示意性实施方案中，物理屏障可位于微流体装置中的分别的区室中，每个区室一个区域。区室可被布置为形成阵列，诸如例如，从Fluidigm Corp.(South SanFrancisco,CA)可得的和本文描述的微流体阵列。还参见图24A-G。In certain embodiments, mechanical capture is used alone or in combination with one or more other capture features to preferentially capture single particles in each respective reaction volume (i.e., each capture site in a microfluidic device). For example, each capture site may include one or more physical barriers sized to contain only one particle. The shape of the physical barrier may be designed to enhance particle retention. For example, when the particles are cells, the size and configuration of the physical barrier may form a concave surface suitable for retaining only one cell. In such embodiments, the physical barrier may be designed to allow fluid to flow through the capture site when it is not occupied by cells, and/or the capture site may include drainage groove features to facilitate this flow. In specific embodiments, the microfluidic device includes multiple appropriately sized/configured physical barriers whereby multiple individual particles are retained in the device, with each physical barrier retaining one particle. In an exemplary embodiment, the physical barriers may be located in separate compartments in the microfluidic device, one area per compartment. The compartments may be arranged to form an array, such as, for example, the microfluidic arrays available from Fluidigm Corp. (South San Francisco, CA) and described herein. See also Figures 24A-G.

在某些实施方案中，基于亲和性的捕获单独使用或与一个或多个其他捕获特征例如机械捕获组合使用以在每个分别的反应体积(即，微流体装置中的每个捕获位点)中优先捕获单细胞。例如，微流体装置表面的包含对颗粒或颗粒组分的结合伴侣的离散区域的尺寸可使得仅一个颗粒可结合该区域，后续颗粒的结合被空间位阻封闭。在具体实施方案中，微流体装置包含多个适当地尺寸的区域，藉以多个单独颗粒被保持在装置中，在每个区域一个颗粒。在示意性实施方案中，这些区域可位于微流体装置中的分别的区室中，每个区室一个区域。区室可被布置为形成阵列，诸如例如，从Fluidigm Corp.(South SanFrancisco,CA)可得的和本文描述的微流体阵列。In certain embodiments, affinity-based capture is used alone or in combination with one or more other capture features, such as mechanical capture, to preferentially capture single cells in each respective reaction volume (i.e., each capture site in a microfluidic device). For example, the size of the discrete regions of the surface of a microfluidic device comprising binding partners for particles or particle components can be such that only one particle can bind to the region, with subsequent particle binding being blocked by steric hindrance. In specific embodiments, the microfluidic device comprises a plurality of appropriately sized regions whereby a plurality of individual particles are retained in the device, one particle in each region. In illustrative embodiments, these regions can be located in separate compartments in the microfluidic device, one region per compartment. The compartments can be arranged to form an array, such as, for example, the microfluidic arrays available from Fluidigm Corp. (South San Francisco, CA) and described herein.

基于亲和性的、优化的单颗粒捕获的一种方法是基于捕获包含结合待检验的颗粒的结合伴侣的支持物。在示意性实施方案中，支持物可以是在其表面分布结合伴侣的珠。参见图23A。珠可通过机械捕获利用杯形捕获特征捕获以在每个捕获位点产生单个固定的支持物(例如，珠)。除了固定支持物以外，在某些实施方案中，捕获特征可减少支持物(例如，珠)展示结合伴侣的表面积。这一表面可被足够地减少使得仅一个颗粒可结合固定的支持物(例如，珠)展示结合伴侣的区域。为了帮助颗粒-支持物结合，在一些实施方案中，固定的支持物展示结合伴侣的区域面对颗粒的流动路径。在具体、示例性的实施方案中，微流体装置的流动通道包含一系列捕获特征。将带有结合伴侣(例如，细胞特异性抗体)的珠的悬液输入通道以在捕获位点产生一系列固定的珠。然后洗涤通道以去除任何游离(即，未固定的)珠。图23A。然后将细胞悬液输入通道。单独细胞可结合每个珠展示结合伴侣的部分。每个结合的细胞经由空间阻塞阻止任何其他细胞结合该珠。通道的洗涤去除未结合的细胞。参见图23B。然后可关闭捕获位点之间的阀以产生分别的反应体积，各自包含一个捕获位点和一个结合的细胞。可采用一个或多个集中特征以引导珠以及颗粒流动朝向每个捕获位点。可选地或另外，捕获特征可各自包括当捕获特征未被珠占据时允许流体流经捕获位点的引流沟特征。One approach to affinity-based, optimized single-particle capture is based on capturing a support containing a binding partner that binds to the particle to be tested. In an illustrative embodiment, the support can be a bead with a binding partner distributed on its surface. See Figure 23A. The beads can be captured by mechanical capture using a cup-shaped capture feature to produce a single fixed support (e.g., bead) at each capture site. In addition to the fixed support, in certain embodiments, the capture feature can reduce the surface area of the support (e.g., bead) displaying the binding partner. This surface can be reduced sufficiently so that only one particle can bind to the area of the fixed support (e.g., bead) displaying the binding partner. To facilitate particle-support binding, in some embodiments, the area of the fixed support displaying the binding partner faces the flow path of the particle. In a specific, exemplary embodiment, the flow channel of the microfluidic device contains a series of capture features. A suspension of beads with a binding partner (e.g., a cell-specific antibody) is introduced into the channel to produce a series of fixed beads at the capture site. The channel is then washed to remove any free (i.e., unfixed) beads. Figure 23A. A cell suspension is then introduced into the channel. Individual cells can bind to the portion of each bead displaying the binding partner. Each bound cell prevents any other cell from binding to the beads via spatial obstruction. The washing of the channel removes unbound cells. Referring to Figure 23 B. The valve between the capture sites can then be closed to produce a respective reaction volume, each comprising a capture site and a bound cell. One or more centralized features can be employed to guide beads and particle flow toward each capture site. Alternatively or in addition, the capture feature can each include a drainage ditch feature that allows fluid to flow through the capture site when the capture feature is not occupied by beads.

确定捕获的颗粒的数目和/或特征Determine the number and/or characteristics of captured particles

在某些实施方案中，确定每个分别的反应体积中颗粒的数目是有利的。当利用有限稀释时，可进行这一确定以鉴定产生具有仅单颗粒的区室的最高比例的稀释。这一确定还可在任何捕获技术之后进行以鉴定包含仅一个颗粒的那些反应体积。例如，在一些实施方案中，基于检验结果是否来自包含0、1、2或更多个细胞的反应体积，可将检验结果分选为多个"仓(bin)"，允许分别分析这些仓的一个或多个。在某些实施方案中，本文描述的任何方法可包括确定任何区室是否包括多于单颗粒(more than a single particle)；和不进一步分析或丢弃来自包括多于单颗粒的任何区室的结果。In certain embodiments, it is advantageous to determine the number of particles in each separate reaction volume. When limiting dilution is utilized, this determination can be made to identify the dilution that produces the highest proportion of compartments with only single particles. This determination can also be made after any capture technique to identify those reaction volumes that contain only one particle. For example, in some embodiments, based on whether the test result is from a reaction volume containing 0, 1, 2 or more cells, the test results can be sorted into multiple "bins", allowing one or more of these bins to be analyzed separately. In certain embodiments, any method described herein can include determining whether any compartment includes more than a single particle; and not further analyzing or discarding the results from any compartment that includes more than a single particle.

在一些实施方案中，每个分别的反应体积中颗粒的数目通过显微术确定。例如，当分别的反应体积是在足够透明或半透明的微流体装置的区室中时，简单的亮视野显微术可用于显现和计数每个区室的颗粒例如细胞。参见实施例5。以下描述的和从Fluidigm Corp.(South San Francisco,CA)可得的微流体装置适于用在这一亮视野显微术方法中。In some embodiments, the number of particles in each separate reaction volume is determined by microscopy. For example, when the separate reaction volumes are in compartments of a sufficiently transparent or translucent microfluidic device, simple bright field microscopy can be used to visualize and count particles, such as cells, in each compartment. See Example 5. The microfluidic device described below and available from Fluidigm Corp. (South San Francisco, CA) is suitable for use in this bright field microscopy method.

在某些实施方案中，可采用染色剂、染料或标记物来检测每个分别的反应体积中颗粒的数目。可使用在分别的反应体积中可检测的任何染色剂、染料或标记物。在示意性实施方案中，可使用荧光染色剂、染料或标记物。采用的染色剂、染料或标记物可为特定应用定制。当颗粒是细胞，且待测量的参数是细胞表面的特征时，染色剂、染料或标记物可以是不需要穿透细胞的细胞表面染色剂、染料或标记物。例如，可采用对细胞表面标志物特异性的标记的抗体来检测每个分别的反应体积中细胞的数目。当颗粒是细胞，且待测量的参数是细胞内部的特征(例如，核酸)时，染色剂、染料或标记物可以是膜透性染色剂、染料或标记物(例如，双链DNA结合染料)。In certain embodiments, staining agent, dyestuff or label can be adopted to detect the number of particle in each reaction volume respectively.Any staining agent, dyestuff or label detectable in the reaction volume respectively can be used.In an illustrative embodiment, fluorescent stain, dyestuff or label can be used.The staining agent, dyestuff or label adopted can be customized for specific application.When particle is cell, and parameter to be measured is the feature of cell surface, staining agent, dyestuff or label can be the cell surface staining agent, dyestuff or label that do not need to penetrate cell.For example, the antibody of the labeling specific to cell surface marker can be adopted to detect the number of cell in each reaction volume respectively.When particle is cell, and parameter to be measured is the feature (for example, nucleic acid) of cell interior, staining agent, dyestuff or label can be membrane permeability staining agent, dyestuff or label (for example, double-stranded DNA binding dye).

在具体实施方案中，在每个分别的反应体积中可检测细胞的特征，伴随或不伴随确定每个反应体积中细胞的数目。例如，可采用染色剂、染料或标记物来确定任何反应体积(例如，微流体装置中的任何区室)是否包括具有该特征的颗粒。这一步骤通过允许后续分析仅包括具有该特定特征的颗粒的那些区室的反应结果可增加检验效率。在这一上下文中可检测的示意性的特征包括，例如，特定基因组重排、拷贝数变化、或多态性；特定基因的表达；和特定蛋白的表达。In a specific embodiment, a characteristic of the cells can be detected in each respective reaction volume, with or without determining the number of cells in each reaction volume. For example, a stain, dye, or marker can be used to determine whether any reaction volume (e.g., any compartment in a microfluidic device) includes particles with that characteristic. This step can increase the efficiency of the assay by allowing subsequent analysis of the reaction results for only those compartments that include particles with that particular characteristic. Illustrative characteristics that can be detected in this context include, for example, specific genomic rearrangements, copy number variations, or polymorphisms; expression of specific genes; and expression of specific proteins.

分析单颗粒中的核酸Analyzing nucleic acids in single particles

在具体实施方案中，本文描述的方法用于分析一个或多个核酸。例如，可确定特定靶核酸的存在和/或水平，也可确定靶核酸的特征，例如，核苷酸序列。在示意性实施方案中，带有在颗粒中或与颗粒缔合的一个或多个样品核酸的颗粒的群体被捕获在分别的反应体积中，分别的反应体积各自优选地包含仅单颗粒。进行反应，诸如连接和/或扩增DNA，或逆转录和/或扩增RNA，对包含一个或多个靶核酸的任何反应体积产生反应产物。这些反应产物可在反应体积中分析，或可分别或以池回收反应体积用于后续分析，诸如DNA测序。In a specific embodiment, the methods described herein are used to analyze one or more nucleic acids. For example, the presence and/or level of a specific target nucleic acid can be determined, and characteristics of the target nucleic acid, such as the nucleotide sequence, can also be determined. In an illustrative embodiment, a population of particles containing one or more sample nucleic acids in or associated with the particles is captured in separate reaction volumes, each of which preferably contains only a single particle. Reactions are performed, such as ligation and/or amplification of DNA, or reverse transcription and/or amplification of RNA, to produce reaction products for any reaction volume containing one or more target nucleic acids. These reaction products can be analyzed in the reaction volume, or the reaction volume can be recovered separately or in a pool for subsequent analysis, such as DNA sequencing.

在某些实施方案中，反应掺入一个或多个核苷酸序列到反应产物中。这些序列可通过任何适合的方法掺入，包括连接、转座酶介导的掺入、或利用带有包括待掺入的序列的一个或多个核苷酸标签的一个或多个引物扩增。这些掺入的核苷酸序列可起到任何帮助本文所述的任何检验的任何功能。例如，一个或多个核苷酸序列可被掺入反应产物以编码有关反应产物的信息条目，诸如为反应产物来源的反应体积的身份。在这种情形中，反应在本文称为"编码反应"。为了这一目的可采用将"条形码"核苷酸序列加入靶核酸的多引物方法并在以上描述。在具体实施方案中，核酸扩增利用至少两个扩增引物进行，其中每个扩增引物包括条形码核苷酸序列，且条形码核苷酸序列的组合编码为反应产物来源的反应体积的身份(称为"组合条形码化")。当分别的反应体积在矩阵型微流体装置的分别的区室中时方便地采用这些实施方案，所述矩阵型微流体装置例如，如从Fluidigm Corp.(South SanFrancisco,CA)可得并在以下描述的那些(参见"微流体装置")。每个分别的区室可包含辨识在其中进行编码反应的区室的行和列的条形码核苷酸序列的组合。如果反应体积被回收并进行包括检测条形码组合的进一步分析，可将结果与特定区室关联，从而与区室中的颗粒关联。这一关联可对包含单颗粒的所有区室进行以允许对颗粒群体的单颗粒(例如，单细胞)分析。In certain embodiments, the reaction incorporates one or more nucleotide sequences into the reaction product. These sequences can be incorporated by any suitable method, including connection, transposase-mediated incorporation or utilizing one or more primer amplifications with one or more nucleotide tags comprising the sequence to be incorporated. The nucleotide sequences of these incorporations can serve any function of any test that helps any of the herein described. For example, one or more nucleotide sequences can be incorporated into the reaction product to encode information items about the reaction product, such as the identity of the reaction volume in the source of the reaction product. In this case, the reaction is referred to as "encoding reaction" herein. For this purpose, a multi-primer method in which a "barcode" nucleotide sequence is added to the target nucleic acid can be adopted and described above. In a specific embodiment, nucleic acid amplification is carried out using at least two amplification primers, wherein each amplification primer includes a barcode nucleotide sequence, and the combination of the barcode nucleotide sequence is encoded as the identity of the reaction volume in the source of the reaction product (referred to as "combination barcoding"). These embodiments are conveniently employed when the separate reaction volumes are in separate compartments of a matrix-type microfluidic device, such as those available from Fluidigm Corp. (South San Francisco, CA) and described below (see "Microfluidic Devices"). Each separate compartment can contain a combination of barcode nucleotide sequences that identify the row and column of the compartment in which the encoded reaction was performed. If the reaction volumes are recovered and subjected to further analysis involving detection of the barcode combination, the results can be associated with a specific compartment, and thus with the particles in the compartment. This association can be performed for all compartments containing a single particle to allow single-particle (e.g., single-cell) analysis of a population of particles.

以下部分讨论适合的核酸样品、和其中适于在本文描述的方法中分析的靶核酸。然后描述扩增引物设计和示例性的扩增方法。其余部分讨论多种标记策略和去除不想要的反应组分。这些部分是关于采用扩增来掺入核酸序列到靶核酸中和/或分析它们的方法来描述的。然而，基于本文的指导，本领域技术人员将认识到，扩增对于进行本文描述的许多方法不是关键的。例如，核酸序列可利用其他手段掺入，诸如连接或利用转座酶。The following sections discuss suitable nucleic acid samples and target nucleic acids suitable for analysis in the methods described herein. Amplification primer design and exemplary amplification methods are then described. The remaining sections discuss various labeling strategies and the removal of unwanted reaction components. These sections describe methods for incorporating nucleic acid sequences into target nucleic acids and/or analyzing them using amplification. However, based on the guidance herein, those skilled in the art will recognize that amplification is not critical for performing many of the methods described herein. For example, nucleic acid sequences can be incorporated using other means, such as ligation or the use of a transposase.

样品核酸Sample nucleic acid

核酸(“样品”)的制备可以从生物来源得到并且使用本领域已知的常规方法进行制备。具体地说，可以从任何来源提取和/或扩增在本文中所述的方法中有用的DNA或RNA，所述来源包括细菌、原生动物、真菌、病毒、细胞器、以及高等生物例如植物类或动物类，具体地是哺乳动物类，并且更具体地是人类。还可以从环境来源(例如，池塘水)，从人造产物(例如，食物)，从法医样品等中得到适当的核酸。可以通过多种标准技术中任何一项从细胞、体液(例如，血液、血液级分、尿液等)、或组织样品中提取或扩增核酸。示例性样品包括血浆、血清、脊髓液、淋巴液、腹膜液、胸膜液、口腔液、以及皮肤的外切片的样品；来自呼吸道、肠道生殖道、以及尿道的样品；眼泪、唾液、血细胞、干细胞、或肿瘤的样品。例如，可以从胚胎中或从母体血液中得到胎儿DNA的样品。可以从活的或死的生物体中或从体外培养物中得到样品。示例性样品可以包括单细胞、福尔马林固定的和/或石蜡包埋的组织样品、以及针吸活组织检查。本文所述的方法中有用的核酸还可以是从一个或多个核酸文库包括cDNA、粘粒、YAC、BAC、PI、PAC文库等中衍生的。Preparation of nucleic acids ("samples") can be obtained from biological sources and prepared using conventional methods known in the art. Specifically, DNA or RNA useful in the methods described herein can be extracted and/or amplified from any source, including bacteria, protozoa, fungi, viruses, organelles, and higher organisms such as plants or animals, specifically mammals, and more specifically humans. Suitable nucleic acids can also be obtained from environmental sources (e.g., pond water), from man-made products (e.g., food), from forensic samples, etc. Nucleic acids can be extracted or amplified from cells, body fluids (e.g., blood, blood fractions, urine, etc.), or tissue samples by any of a variety of standard techniques. Exemplary samples include samples of plasma, serum, spinal fluid, lymph fluid, peritoneal fluid, pleural fluid, oral fluid, and external sections of skin; samples from the respiratory tract, intestinal genital tract, and urinary tract; samples of tears, saliva, blood cells, stem cells, or tumors. For example, a sample of fetal DNA can be obtained from an embryo or from maternal blood. Samples can be obtained from living or dead organisms or from in vitro cultures. Exemplary samples can include single cells, formalin-fixed and/or paraffin-embedded tissue samples, and needle biopsies. The nucleic acids useful in the methods described herein can also be derived from one or more nucleic acid libraries, including cDNA, cosmid, YAC, BAC, PI, PAC libraries, etc.

可以使用本领域熟知的方法分离感兴趣的核酸，其中具体方法的选择取决于核酸的来源、性质、以及类似因素。这些样品核酸不必是纯的形式，但是典型地是足够纯的以允许目标反应得以实施。当靶核酸是RNA时，该RNA可以通过本领域中已知的并且在例如Sambrook,J.,Fritsch,E.F.,和Maniatis,T.,Molecular Cloning:A LaboratoryManual.Cold Spring Harbor Laboratory Press,NY,Vol.1,2,3(1989)中进行说明的标准方法被逆转录成为cDNA。Nucleic acids of interest can be isolated using methods well known in the art, wherein the selection of a specific method depends on the source, properties, and similar factors of the nucleic acid. These sample nucleic acids need not be in pure form, but are typically sufficiently pure to allow the target reaction to be implemented. When the target nucleic acid is RNA, the RNA can be reverse transcribed into cDNA by standard methods known in the art and described in, for example, Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989).

靶核酸Target nucleic acid

在本文所述的方法中有用的靶核酸可以从以上所述的任何样品核酸衍生。在典型的实施方案中，对于靶核酸而言，至少一些核苷酸序列信息应该是已知的。例如，如果使用PCR作为编码反应，总体上对于给定的靶核酸的每个末端而言可以得到足够的序列信息以允许设计适当的扩增引物。在替代实施方案中，引物中靶特异性序列可以用随机或简并的核苷酸序列来替换。In the methods described herein, useful target nucleic acids can be derived from any sample nucleic acid described above. In typical embodiments, at least some nucleotide sequence information should be known for the target nucleic acid. For example, if PCR is used as an encoding reaction, generally enough sequence information can be obtained for each end of a given target nucleic acid to allow for the design of appropriate amplification primers. In alternative embodiments, the target-specific sequence can be replaced with a random or degenerate nucleotide sequence in the primer.

这些靶可以包括例如与病原体例如病毒、细菌、原生动物、或真菌相关的核酸；RNA，例如，过表达或低表达指示疾病的那些，以组织特异性或发育特异性形式表达的那些；或由特定的刺激物诱导的那些；基因组DNA，该基因组DNA可以被分析特异性多态性(例如SNP)、等位基因、或单体型，例如在基因分型中。特别感兴趣的是在遗传疾病或其他病理学中发生改变的(例如，扩增、缺失、重排、和/或突变)基因组DNA；与希望的或不希望的性状相关联的序列；和/或独特地识别个体的序列(例如，在法医或亲子鉴定中)。当采用多个靶核酸时，这些可以在相同的或不同的染色体上。These targets can include, for example, nucleic acids associated with pathogens such as viruses, bacteria, protozoa, or fungi; RNA, for example, those that overexpress or underexpress indicate a disease, those expressed in a tissue-specific or development-specific form; or those induced by a specific stimulus; genomic DNA, which can be analyzed for specific polymorphisms (e.g., SNPs), alleles, or haplotypes, such as in genotyping. Of particular interest are genomic DNAs that are altered (e.g., amplified, deleted, rearranged, and/or mutated) in genetic diseases or other pathologies; sequences associated with desired or undesirable traits; and/or sequences that uniquely identify an individual (e.g., in forensic or paternity testing). When multiple target nucleic acids are employed, these can be on the same or different chromosomes.

在多个实施方案中，待扩增的靶核酸可以是，例如，25个碱基、50个碱基、100个碱基、200个碱基、500个碱基或750个碱基。在本文描述的方法的某些实施方案中，可采用长片段(long-range)扩增方法诸如长片段PCR以从扩增混合物产生扩增子。长片段PCR允许扩增范围从1或数千碱基(kb)至超过50kb的靶核酸。在多个实施方案中，通过长片段PCR扩增的靶核酸的长度是至少约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25、30、35、40、45或50kb。靶核酸还可落入以这些数值的任一个作为端点的任何范围内(例如，25个碱基至100个碱基或5-15kb)。In some embodiments, the target nucleic acid to be amplified can be, for example, 25 bases, 50 bases, 100 bases, 200 bases, 500 bases or 750 bases. In some embodiments of the methods described herein, long-range amplification methods such as long-range PCR can be adopted to produce amplicon from amplification mixture. Long-range PCR allows amplification range from 1 or several thousand bases (kb) to a target nucleic acid exceeding 50kb. In some embodiments, the length of the target nucleic acid amplified by long-range PCR is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50kb. Target nucleic acid can also fall into any range (for example, 25 bases to 100 bases or 5-15kb) with any one of these numerical values as endpoint.

引物设计Primer design

适合用于核酸扩增的引物是足够长的以在用于聚合的试剂存在下引发延伸产物的合成。引物的确切长度以及构成将取决于多种因素，包括例如退火反应的温度、引物的来源以及构成，以及当使用探针时，探针退火位点与引物退火位点的接近程度以及引物:探针浓度的比率。例如，取决于靶核酸序列的复杂性，寡核苷酸引物典型地包含在约15至约30个核苷酸的范围内，虽然它可以包含更多或更少的核苷酸。引物应当是足够互补的以选择性地退火到它们相应的链上并且形成稳定的双链体。本领域普通技术人员知道如何选择适当的引物对来扩增感兴趣的靶核酸。The primer that is suitable for nucleic acid amplification is long enough to initiate the synthesis of extension products in the presence of a reagent for polymerization. The exact length of primer and formation will depend on multiple factors, including the temperature of the annealing reaction, the source of primer and formation, and when using probe, the proximity of the probe annealing site and the primer annealing site and the ratio of primer: probe concentration. For example, depending on the complexity of the target nucleic acid sequence, oligonucleotide primers are typically included in the scope of approximately 15 to approximately 30 nucleotides, although it can comprise more or less nucleotides. Primers should be sufficiently complementary to selectively anneal to their corresponding chain and form stable duplexes. Those of ordinary skill in the art know how to select suitable primers to increase interested target nucleic acid.

例如，可以通过使用任何可商购的软件或开源软件例如Primer3(参见，例如，Rozen和Skaletsky(2000)Meth.Mol.Biol,132:365-386；www.broad.mit.edu/node/1060，等)或者通过访问Roche UPL网址来设计PCR引物。将扩增子序列输入Primer3程序中，其中UPL探针序列在括号中以确保Primer3程序将在被括起来的探针序列的任一侧上设计引物。For example, PCR primers can be designed by using any commercially available software or open source software such as Primer3 (see, e.g., Rozen and Skaletsky (2000) Meth. Mol. Biol, 132:365-386; www.broad.mit.edu/node/1060, etc.) or by accessing the Roche UPL website. The amplicon sequence is entered into the Primer3 program with the UPL probe sequence in brackets to ensure that the Primer3 program will design primers on either side of the bracketed probe sequence.

可以通过任何适当的方法来制备引物，包括例如，适当序列的克隆以及限制性酶切或通过多种方法的直接化学合成，例如Narang等人.(1979)Meth.Enzymol.68:90-99的磷酸三酯法；Brown等人(1979)Meth.Enzymol.68:109-151的磷酸二酯法；Beaucage等人(1981)Tetra.Lett.,22:1859-1862的二乙基磷酰胺酸法；美国专利号4,458,066的固体载体法等，或可以从商业来源提供引物。Primers can be prepared by any suitable method, including, for example, cloning of appropriate sequences and restriction enzyme digestion or direct chemical synthesis by a variety of methods, such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109-151; the diethylphosphamidate method of Beaucage et al. (1981) Tetra. Lett., 22:1859-1862; the solid support method of U.S. Pat. No. 4,458,066, etc., or primers can be provided from commercial sources.

可以通过使用Sephadex柱(Amersham Biosciences,Inc.,Piscataway,NJ)或本领域普通技术人员已知的其他方法对引物进行纯化。引物纯化可以提高本发明方法的灵敏度。Primers can be purified by using Sephadex columns (Amersham Biosciences, Inc., Piscataway, NJ) or other methods known to those of ordinary skill in the art. Primer purification can improve the sensitivity of the method of the present invention.

扩增方法Amplification method

核酸可根据本文描述的方法为了任何有用的目的扩增，例如，以增加靶核酸的浓度用于后续分析、和/或掺入一个或多个核苷酸序列、和/或检测和/或定量和/或测序一个或多个靶核酸。扩增可在液滴中、在乳液中、在容器中、在微量滴定板的孔中、在矩阵型微流体装置的区室、等等中进行。Nucleic acids can be amplified according to the methods described herein for any useful purpose, for example, to increase the concentration of target nucleic acids for subsequent analysis, and/or to incorporate one or more nucleotide sequences, and/or to detect and/or quantify and/or sequence one or more target nucleic acids. Amplification can be performed in droplets, in emulsions, in containers, in wells of microtiter plates, in compartments of matrix-type microfluidic devices, and the like.

扩增以增加靶核酸浓度Amplification to increase target nucleic acid concentration

扩增以增加靶核酸浓度可旨在扩增反应混合物中所有核酸、特定类型的所有核酸(例如，DNA或RNA)、或特定靶核酸。在具体、示例性的实施方案中，可进行全基因组扩增以增加基因组DNA的浓度；可扩增RNA，任选地先进行逆转录步骤；和/或一般性或靶特异性预扩增。Amplification to increase the concentration of target nucleic acids can be intended to amplify all nucleic acids in the reaction mixture, all nucleic acids of a particular type (e.g., DNA or RNA), or a specific target nucleic acid. In specific, exemplary embodiments, whole genome amplification can be performed to increase the concentration of genomic DNA; RNA can be amplified, optionally preceded by a reverse transcription step; and/or general or target-specific pre-amplification can be performed.

全基因组扩增Whole genome amplification

为分析基因组DNA，可以使用全基因组扩增(WGA)方法扩增样品核酸。合适的WGA的方法包括引物延伸PCR(PEP)和改进的PEP(I-PEP)、简并寡核苷酸引物PCR(DOP-PCR)、连接介导PCR(LMP)、基于T7的DNA线性扩增(TLAD)、和多重置换扩增(MDA)。这些技术描述在2010年7月15日公布的美国专利公布号20100178655中(Hamilton等人)，其通过引用全文并入本文，尤其是其可用于单细胞核酸分析的方法的描述。For analyzing genomic DNA, whole genome amplification (WGA) method can be used to amplify sample nucleic acid. Suitable WGA methods include primer extension PCR (PEP) and improved PEP (I-PEP), degenerate oligonucleotide primer PCR (DOP-PCR), connection-mediated PCR (LMP), DNA linear amplification (TLAD) based on T7, and multiple displacement amplification (MDA). These techniques are described in U.S. Patent Publication No. 20100178655 (Hamilton et al.), published on July 15, 2010, which is incorporated herein by reference in its entirety, and especially the description of the method for single cell nucleic acid analysis.

WGA试剂盒可购自例如，Qiagen,Inc.(Valencia,CA USA)、Sigma-Aldrich(Rubicon Genomics；例如，SigmaSingle Cell Whole GenomeAmplification Kit,PN WGA4-50RXN)。本文描述的方法的WGA步骤可以使用任何可利用的试剂盒根据生产商的说明而进行。WGA kits are available from, for example, Qiagen, Inc. (Valencia, CA USA), Sigma-Aldrich (Rubicon Genomics; for example, Sigma Single Cell Whole Genome Amplification Kit, PN WGA4-50RXN). The WGA step of the methods described herein can be performed using any available kit according to the manufacturer's instructions.

在具体实施方案中，WGA步骤是限制的WGA，即，WGA在达到反应平台期之前停止。典型地，WGA进行多于两个扩增循环。在某些实施方案中，WGA进行少于大约10个扩增循环，例如4至8个循环之间，包括4和8个循环。但是，WGA可以进行3、4、5、6、7、8或9个循环，或者落入由任意这些值限定的范围内的一些循环。In a specific embodiment, the WGA step is a restricted WGA, that is, WGA stops before reaching the reaction plateau. Typically, WGA carries out more than two amplification cycles. In certain embodiments, WGA carries out less than about 10 amplification cycles, for example, between 4 to 8 cycles, including 4 and 8 cycles. However, WGA can carry out 3, 4, 5, 6, 7, 8 or 9 cycles, or fall into some cycles within the scope limited by any of these values.

RNA扩增RNA amplification

在某些实施方案中，可以分析单细胞或小细胞群的RNA中的一个或多个RNA靶。合适的RNA靶包括mRNA，以及非编码RNA，如小核仁RNA(snoRNA)、微小RNA(miRNA)、小干扰RNA(siRNA)和与Piwi蛋白相作用的RNA(Piwi-interacting RNA)(piRNA)。在具体实施方案中，感兴趣的RNA被转化成DNA，如通过逆转录或扩增。In certain embodiments, one or more RNA targets can be analyzed in the RNA of a single cell or a small population of cells. Suitable RNA targets include mRNA, as well as non-coding RNAs such as small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), and RNA that interacts with Piwi proteins (Piwi-interacting RNA) (piRNA). In specific embodiments, the RNA of interest is converted to DNA, such as by reverse transcription or amplification.

例如，为分析单细胞或小细胞群的mRNA，所述mRNA通常被转化成mRNA群的DNA表现形式。在某些实施方案中，使用的方法优选地产生cDNA群，其中每一个cDNA的相对量与样品群中对应的mRNA的相对量大致相同。For example, to analyze the mRNA of a single cell or a small cell population, the mRNA is typically converted into a DNA representation of the mRNA population. In certain embodiments, the method used preferably produces a cDNA population in which the relative amount of each cDNA is approximately the same as the relative amount of the corresponding mRNA in the sample population.

在具体实施方案中，可以采用逆转录根据标准方法使用逆转录酶从mRNA模板产生cDNA。通过使用如特异引物、寡聚dT或随机引物可以启动细胞mRNA群的逆转录。为合成代表细胞mRNA的cDNA库，可以使用逆转录酶合成与样品细胞RNA互补的cDNA第一链。这可以使用商业可利用的BRL Superscript II试剂盒(BRL,Gaithersburg,Md.)或任何其它商业可利用的试剂盒完成。逆转录酶优先使用RNA作为模板，但也可以使用单链DNA模板。因此，可以使用逆转录酶和合适的引物(如，多聚A、随机引物等等)完成cDNA第二链的合成。还可以使用大肠杆菌(E.coli)DNA聚合酶I完成第二链合成。可以在第二cDNA链合成的同时或之后移除所述RNA。这可以通过例如用降解RNA的RNA酶如大肠杆菌RNA酶H处理混合物而完成。In a specific embodiment, reverse transcription can be adopted to produce cDNA from mRNA template using reverse transcriptase according to standard methods. By using, for example, specific primers, oligo-dT or random primers, reverse transcription of a cell mRNA group can be initiated. For synthesizing a cDNA library representing cell mRNA, reverse transcriptase can be used to synthesize a first cDNA chain complementary to the sample cell RNA. This can be accomplished using commercially available BRL Superscript II test kit (BRL, Gaithersburg, Md.) or any other commercially available test kit. Reverse transcriptase preferentially uses RNA as a template, but single-stranded DNA templates can also be used. Therefore, reverse transcriptase and suitable primers (e.g., poly A, random primers, etc.) can be used to complete the synthesis of the cDNA second chain. Escherichia coli (E. coli) DNA polymerase I can also be used to complete the synthesis of the second chain. The RNA can be removed while or after the second cDNA chain is synthesized. This can be accomplished by, for example, treating the mixture with an RNA enzyme such as E. coli RNase H that degrades RNA.

在其他实施方案中，使用扩增方法从mRNA模板产生cDNA。在这样的实施方案中，典型地使用产生代表mRNA群的cDNA群的扩增方法。In other embodiments, amplification methods are used to generate cDNA from an mRNA template. In such embodiments, amplification methods that generate a cDNA population representative of an mRNA population are typically used.

单细胞或小细胞群的非编码RNA的分析典型地起始于感兴趣的RNA转化成DNA。该转化可以通过逆转录或扩增完成。在某些实施方案中，使用的方法优选地产生DNA群，其中每一个DNA的相对量与样品群中对应的mRNA的相对量大致相同。可以使用优先与感兴趣的RNA退火的引物选择性地逆转录或扩增所述靶RNA。合适的引物可以商业获得或可由本领域技术人员设计。例如，Life Technologies出售用于微小RNA(miRNA)靶的MegaPlex^TM引物池。这些引物可以用于逆转录(RT)和特定靶扩增(STA)。参见，如实施例6B。The analysis of the non-coding RNA of a single cell or small cell group typically begins with the conversion of the RNA of interest into DNA. This conversion can be completed by reverse transcription or amplification. In certain embodiments, the method used preferably produces a DNA group, wherein the relative amount of each DNA is roughly the same as the relative amount of the corresponding mRNA in the sample group. Primers that preferentially anneal to the RNA of interest can be used to selectively reverse transcribe or amplify the target RNA. Suitable primers can be commercially available or can be designed by those skilled in the art. For example, Life Technologies sells MegaPlex^™ primer pools for microRNA (miRNA) targets. These primers can be used for reverse transcription (RT) and specific target amplification (STA). See, for example, Example 6B.

预扩增Pre-amplification

可进行预扩增来增加反应混合物中核酸序列的浓度，一般，例如利用随机引物组、对存在的多个或所有核酸共同的一个或多个序列特异性的引物(例如，多聚-dT来引发多聚-A尾)、或随机引物组和特异性引物的组合。可选地，预扩增可利用对感兴趣的一个或多个靶核酸特异性的一个或多个引物对进行。在特定、示例性实施方案中，可预扩增由WGA产生的扩增的基因组或从RNA产生的DNA(如cDNA)以产生预扩增反应混合物，所述预扩增反应混合物包含一个或多个特异于一个或多个感兴趣的靶核酸的扩增子。典型地，使用预扩增引物、合适的缓冲液系统、核苷酸和DNA聚合酶(如修改为用于“热启动”条件的聚合酶)进行预扩增。Pre-amplification can be carried out to increase the concentration of nucleic acid sequence in the reaction mixture, generally, for example, using random primer sets, primers specific to one or more sequences common to multiple or all nucleic acids present (for example, poly-dT triggers poly-A tails) or a combination of random primer sets and specific primers. Alternatively, pre-amplification can be carried out using one or more primers specific to one or more target nucleic acids of interest. In specific, exemplary embodiments, the genome of the amplification produced by WGA or the DNA (such as cDNA) produced from RNA can be pre-amplified to produce a pre-amplification reaction mixture, which comprises one or more amplicons specific to one or more target nucleic acids of interest. Typically, pre-amplification is carried out using pre-amplification primers, a suitable buffer system, nucleotides and an archaeal dna polymerase (such as a polymerase modified for "hot start" conditions).

在具体实施方案中，预扩增引物是与在制备样品的扩增实验中使用的那些相同的序列，尽管通常是以减少的浓度。引物浓度可以是如比在扩增实验中使用的引物浓度少大约10至大约250倍。实施方案包括使用比在扩增实验中的引物浓度少大约10、20、35、50、65、75、100、125、150、175和200倍的引物。In a specific embodiment, the pre-amplification primers are the same sequences as those used in the amplification experiment to prepare the sample, although typically at a reduced concentration. The primer concentration can be, for example, about 10 to about 250 times less than the primer concentration used in the amplification experiment. Embodiments include using primers that are about 10, 20, 35, 50, 65, 75, 100, 125, 150, 175, and 200 times less than the primer concentration in the amplification experiment.

在具体实施方案中，预扩增至少进行两个循环。在某些实施方案中，预扩增进行少于大约20个循环，如8和18个循环之间，包括8和18个循环。但是，预扩增可以进行3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24个循环或者落入任何这些值限定的范围内的一些循环。在示例的实施方式中，预扩增可以进行大约14个循环以将被检测的扩增子增加大约16,000倍。In a specific embodiment, pre-amplification carries out at least two circulations.In certain embodiments, pre-amplification carries out less than about 20 circulations, as between 8 and 18 circulations, comprise 8 and 18 circulations.But pre-amplification can carry out 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 or 24 circulations or fall into some circulations in the scope of any these value limits.In the embodiment of example, pre-amplification can carry out about 14 circulations to increase the amplicon to be detected by about 16,000 times.

扩增用于检测和/或定量靶核酸Amplification for detection and/or quantification of target nucleic acids

在本文所述的方法中可以使用核酸检测和/或定量的任何方法以检测扩增产物。在一个实施方案中，使用PCR(聚合酶链式反应)来对靶核酸进行扩增和/或定量。在其他实施方案中，使用其他扩增系统或检测系统，包括例如美国专利号7,118,910中所述的系统(出于它的说明扩增/检测系统的目的，该专利通过引用以其全部内容并入本文)。在具体实施方案中，使用实时定量的方法。例如可以使用“定量实时PCR”方法通过测量在扩增过程本身期间形成的扩增产物的数量来确定样品中存在的靶核酸的量。In the methods described herein, nucleic acid detection and/or quantitative any method can be used to detect amplified product. In one embodiment, PCR (polymerase chain reaction) is used to increase and/or quantitatively target nucleic acid. In other embodiments, other amplification systems or detection systems are used, including for example the system described in U.S. Patent number 7,118,910 (for the purpose of its explanation amplification/detection system, this patent is incorporated herein by reference in its entirety). In a specific embodiment, the method for real-time quantitative detection is used. For example, " quantitative real-time PCR " method can be used to determine the amount of the target nucleic acid present in the sample by measuring the quantity of the amplified product formed during the amplification process itself.

荧光生成核酸酶测定法(fluorogenic nuclease assay)是实时定量方法的一个具体的实例，该方法可以被成功地用于在本文所述的方法中。监测扩增产物形成的方法包括使用双标记的荧光生成寡核苷酸探针对PCR产物累积进行连续测量，文献中通常称为“方法”的一种方法。参见美国专利号5,723,591；Heid等人,1996,Real-timequantitative PCR Genome Res.6:986-94，出于它们的说明荧光生成核酸酶测定法的目的，每个文件都通过引用以其全部内容并入本文。应当理解的是虽然对于qPCR而言“探针”是最广泛使用的，本文所述的方法不限于使用这些探针；可以使用任何适合的探针。Fluorescence generation nuclease assay (fluorogenic nuclease assay) is a specific example of a real-time quantitative method, which can be successfully used in the method described herein. The method for monitoring the formation of amplified products includes continuously measuring the accumulation of PCR products using a double-labeled fluorescence generation oligonucleotide probe, a method generally referred to as a "method" in the literature. See U.S. Patent No. 5,723,591; Heid et al., 1996, Real-time quantitative PCR Genome Res. 6:986-94, for the purpose of their description of the fluorescence generation nuclease assay method, each of which is incorporated herein by reference in its entirety. It should be understood that although "probe" is the most widely used for qPCR, the methods described herein are not limited to the use of these probes; any suitable probe can be used.

可以用于本发明的其他检测/定量方法包括FRET以及模板延伸反应、分子信标检测、蝎型探针检测(scorpion detection)、侵入物检测(invader detection)、以及锁式探针检测(padlock probe detection)。Other detection/quantification methods that can be used in the present invention include FRET and template extension reactions, molecular beacon detection, scorpion detection, invader detection, and padlock probe detection.

FRET和模板延伸反应使用用供体/受体对的一个成员标记的引物以及用该供体/受体对的另一个成员标记的核苷酸。在模板依赖性延伸反应期间在将标记的核苷酸掺入到引物中之前，供体和受体被间隔足够开这样使得能量转移不会发生。然而，如果标记的核苷酸被掺入到引物中并且该间距是足够近的，则发生能量转移并且可以被检出。这些方法在检测单核苷酸多态性中在进行单碱基对延伸反应中是特别有用的，并且在美国专利号5,945,283以及PCT公布WO 97/22719中描述。In some embodiments, the nucleotide sequence of the present invention is nucleotide sequence.FRET and template extension reaction use the primer of a member mark of donor/acceptor pair and the Nucleotide of another member mark of this donor/acceptor pair.During the template dependency extension reaction, before the Nucleotide of mark was incorporated in the primer, donor and acceptor were spaced enough to open so that energy transfer can not take place.Yet, if the Nucleotide of mark is incorporated in the primer and this spacing is close enough, then energy transfer takes place and can be detected.These methods are particularly useful in carrying out the single base pair extension reaction in detecting SNP, and are described in U.S. Patent number 5,945,283 and PCT publication WO 97/22719.

关于分子信标，当探针杂交到扩增产物的互补区上时，它的构象改变导致形成可检出的信号。探针本身包括两个区段：一个区段在5’末端并且另一个区段在3’末端。这些区段位于退火到探针结合位点的探针区段的侧翼，并且彼此互补。一个末端区段被典型地附加到报告物染料(reporter dye)上，并且另一个末端区段通常被附连到猝灭剂染料(quencher dye)上。在溶液中，两个末端区段可以彼此杂交从而形成发夹环。在这种构象中，报告物和猝灭剂染料是足够近的接近这样使得来自报告物染料的荧光被猝灭剂染料有效地猝灭。相反，杂交的探针导致其中猝灭程度被降低的线性构象。因此，通过监测这两种染料的发射变化，有可能间接地监测扩增产物的形成。这个类型的探针以及它们的使用方法进一步由例如Piatek等人,1998,Nat.Biotechnol.16:359-63；Tyagi,和Kramer,1996,Nat.Biotechnology14:303-308；和Tyagi,等人,1998,Nat.Biotechnol.16:49-53(1998)描述。About molecular beacons, when the probe hybridizes to the complementary region of the amplified product, its conformational change causes the formation of a detectable signal. The probe itself includes two segments: one segment at the 5' end and the other segment at the 3' end. These segments are located on the flanks of the probe segment that anneals to the probe binding site and are complementary to each other. One end segment is typically attached to a reporter dye, and the other end segment is usually attached to a quencher dye. In solution, the two end segments can hybridize with each other to form a hairpin loop. In this conformation, the reporter and the quencher dye are close enough so that the fluorescence from the reporter dye is effectively quenched by the quencher dye. In contrast, the hybridized probe causes a linear conformation in which the degree of quenching is reduced. Therefore, by monitoring the emission changes of these two dyes, it is possible to indirectly monitor the formation of the amplified product. Probes of this type and methods for their use are further described by, for example, Piatek et al., 1998, Nat. Biotechnol. 16:359-63; Tyagi, and Kramer, 1996, Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat. Biotechnol. 16:49-53 (1998).

蝎型探针检测方法由例如Thelwell等人.2000,Nucleic Acids Research,28:3752-3761和Solinas等人,2001,"Duplex Scorpion primers in SNP analysis and FRETapplications"Nucleic Acids Research 29:20描述。蝎型引物是荧光生成PCR引物，其中探针元件经由PCR终止剂(PCR stopper)被附加到5’端上。它们被用于均匀溶液中PCR产物的实时扩增子特异性检测。两种不同形式是可能的，即“茎环”形式和“双链体”形式。在两种情况中，探查机理是分子内的。在所有形式中蝎型探针检测的基本元件是：(i)PCR引物；(ii)用于防止PCR读通该探针元件的PCR终止剂；(iii)特异性探针序列；以及(iv)包含至少一个荧光团以及猝灭剂的荧光检测系统。在蝎型引物PCR延伸之后，得到的扩增子包含与探针互补的序列，这导致了每个PCR循环的变性阶段形成单链。当冷却时，探针是游离的以结合到这个互补序列上，造成荧光增加，因为猝灭剂不再位于该荧光团的附近。PCR终止剂防止探针被Taq DNA聚合酶不希望地读通。The scorpion probe detection method is described, for example, by Thelwell et al. 2000, Nucleic Acids Research, 28:3752-3761 and Solinas et al. 2001, "Duplex Scorpion primers in SNP analysis and FRET applications" Nucleic Acids Research 29:20. Scorpion primers are fluorescence-generating PCR primers in which the probe element is attached to the 5' end via a PCR stopper. They are used for real-time amplicon-specific detection of PCR products in homogeneous solution. Two different formats are possible, namely the "stem-loop" format and the "duplex" format. In both cases, the detection mechanism is intramolecular. The essential elements of scorpion probe detection in all formats are: (i) a PCR primer; (ii) a PCR terminator to prevent PCR read-through of the probe element; (iii) a specific probe sequence; and (iv) a fluorescence detection system comprising at least one fluorophore and a quencher. After PCR extension with the Scorpion primers, the resulting amplicon contains a sequence complementary to the probe, which results in single-stranded formation during the denaturation phase of each PCR cycle. Upon cooling, the probe is free to bind to this complementary sequence, resulting in increased fluorescence because the quencher is no longer in the vicinity of the fluorophore. PCR terminators prevent the probe from being undesirably read through by Taq DNA polymerase.

侵入物测定法(Third Wave Technologies,Madison,WI)特别地用于SNP基因分型并且使用称为信号探针的寡核苷酸，该寡核苷酸是与靶核酸(DNA或RNA)或多态性位点互补的。称为Invader Oligo的第二寡核苷酸包含相同的5’核苷酸序列，但是3’核苷酸序列包含核苷酸多态性。Invader Oligo干扰信号探针结合到靶核酸上，这样使得信号探针的5’端在包含该多态性的核苷酸上形成一个“悬垂片”。这个复合体被称为裂解酶(cleavase)的结构特异性内切核酸酶所识别。裂解酶裂解核苷酸的5’悬垂片。释放的悬垂片与携带FRET标记物的第三探针相结合，由此形成被裂解酶所识别的另一个双链体结构。这次该裂解酶裂解荧光团远离猝灭剂并且生成荧光信号。对于SNP基因分型而言，将设计信号探针以与参考(野生型)等位基因或变异体(突变体)等位基因之一相杂交。与PCR不同，存在信号的线性扩增而没有核酸的扩增。通过下面各项提供了足以指导本领域普通技术人员的进一步的细节，例如Neri,B.P.,等人,Advances in Nucleic Acid and Protein Analysis 3826:117-125,2000)以及美国专利号6,706,471。Invader assay (Third Wave Technologies, Madison, WI) is particularly used for SNP genotyping and uses oligonucleotides called signal probes that are complementary to target nucleic acids (DNA or RNA) or polymorphic sites. A second oligonucleotide called Invader Oligo contains the same 5' nucleotide sequence, but the 3' nucleotide sequence contains a nucleotide polymorphism. The Invader Oligo interference signal probe is bound to the target nucleic acid so that the 5' end of the signal probe forms an "overhang" on the nucleotide containing the polymorphism. This complex is recognized by a structure-specific endonuclease called a cleavage enzyme. The cleavage enzyme cleaves the 5' overhang of the nucleotide. The released overhang is combined with a third probe carrying a FRET marker to form another duplex structure recognized by the cleavage enzyme. This time, the cleavage enzyme cleaves the fluorophore away from the quencher and generates a fluorescent signal. For SNP genotyping, a signal probe is designed to hybridize with one of the reference (wild-type) alleles or variant (mutant) alleles. Unlike PCR, there is linear amplification of the signal without amplification of the nucleic acid. Further details sufficient to guide one of ordinary skill in the art are provided by, for example, Neri, B.P., et al., Advances in Nucleic Acid and Protein Analysis 3826:117-125, 2000) and U.S. Patent No. 6,706,471.

锁式探针(PLP)是长的(例如，约100个碱基)线性寡核苷酸。在探针的3’和5’末端上的序列是与靶核酸中邻近序列互补的。在PLP的中央非互补区中，存在可以被用于识别特异性PLP的“标签”序列。标签序列位于通用引发位点侧翼，引发位点允许PCR扩增该标签。当杂交到靶上时，PLP寡核苷酸的两个末端变成紧密接近并且可以通过酶连接作用进行连接。得到的产物是连锁(catenated)到靶DNA链上的环形探针分子。通过核酸外切酶的作用去除任何未连接的探针(即没有杂交到靶上的探针)。PLP的杂交作用和连接要求两个末端区段都识别靶序列。以这种方式，PLP提供了极其特异性的靶识别。Padlock probes (PLPs) are long (e.g., about 100 bases) linear oligonucleotides. The sequences at the 3' and 5' ends of the probe are complementary to adjacent sequences in the target nucleic acid. In the central non-complementary region of the PLP, there is a "tag" sequence that can be used to identify a specific PLP. The tag sequence flanks a universal priming site that allows PCR amplification of the tag. When hybridized to the target, the two ends of the PLP oligonucleotide become closely adjacent and can be connected by enzyme ligation. The resulting product is a circular probe molecule catenated to the target DNA chain. Any unconnected probe (i.e., a probe that is not hybridized to the target) is removed by the action of an exonuclease. The hybridization and connection of the PLP require that both end segments recognize the target sequence. In this way, PLP provides extremely specific target recognition.

然后可以将环化PLP的标签区扩增并且对得到的扩增子进行检测。例如，可以进行实时PCR以对扩增子进行检测和定量。扩增子的存在和数量可能与样品中靶序列的存在和数量相关联。关于PLP的说明，参见例如，Landegren等人,2003,Padlock andproximity probes for in situ and array-based analyses:tools for the post-genomic era,Comparative and Functional Genomics 4:525-30；Nilsson等人,2006,Analyzing genes using closing and replicating circles Trends BiotechnoX.24:83-8；Nilsson等人,1994,Padlock probes:circularizing oligonucleotides forlocalized DNA detection,Science 265:2085-8。The tag region of the circularized PLP can then be amplified and the resulting amplicon detected. For example, real-time PCR can be performed to detect and quantify the amplicon. The presence and amount of the amplicon may be correlated with the presence and amount of the target sequence in the sample. For a description of PLP, see, for example, Landegren et al., 2003, Padlock and proximity probes for in situ and array-based analyses: tools for the post-genomic era, Comparative and Functional Genomics 4: 525-30; Nilsson et al., 2006, Analyzing genes using closing and replicating circles, Trends BiotechnoX. 24: 83-8; Nilsson et al., 1994, Padlock probes: circularizing oligonucleotides for localized DNA detection, Science 265: 2085-8.

在具体实施方案中，可以用作针对探针的检测标记物的荧光团包括但不限于罗丹明、菁蓝3(Cy3)、菁蓝5(Cy5)、荧光素、Vic^TM、LiZ^TM、Tamra^TM、5-Fam^TM、6-Fam^TM、以及德克萨斯红(Molecular Probes)。(Vic^TM、LiZ^TM、Tamra^TM、5-Fam^TM、6-Fam^TM都是从LifeTechnologies,Foster City,Calif可得到的)。In specific embodiments, fluorophores that can be used as detection labels for the probe include, but are not limited to, rhodamine, cyanine 3 (Cy3), cyanine 5 (Cy5), fluorescein, Vic^™ , LiZ^™ , Tamra^™ , 5-Fam^™ , 6-Fam^™ , and Texas Red (Molecular Probes). (Vic^™ , LiZ^™ , Tamra^™ , 5-Fam^™ , 6-Fam^™ are all available from Life Technologies, Foster City, Calif.).

在一些实施方案中，在足以表明样品中存在靶核酸序列的预定数目的循环之后技术人员可以简单地进行对扩增产物的量监测。对于任何给定的样品类型、引物序列、以及反应条件，本领域技术人员可以容易地确定多少个循环对于确定给定靶核酸的存在是足够的。在其他实施方案中，在指数扩增结束时，即在“平台”期期间进行检测，或进行终点PCR。在不同实施方案中，扩增可以被执行约：2个、4个、10个、15个、20个、25个、30个、35个或40个循环或落在由这些值的任何一项界定的任何范围内的数量的循环。In some embodiments, after the predetermined number of cycles that are enough to show that there is target nucleic acid sequence in the sample, technicians can simply carry out the amount monitoring of amplified product.For any given sample type, primer sequence and reaction conditions, those skilled in the art can easily determine how many cycles are enough for determining the existence of given target nucleic acid.In other embodiments, when exponential amplification finishes, promptly detect during the " platform " phase, or carry out endpoint PCR.In different embodiments, amplification can be performed approximately: 2,4,10,15,20,25,30,35 or 40 cycles or fall on the cycle of the quantity in any scope defined by any one of these values.

通过获得在不同温度下的荧光，可能追踪杂交的程度。此外，PCR产物杂交的温度依赖性可以用于鉴定和/或定量PCR产物。因此，本文描述的方法包括解链曲线分析在检测和/或定量扩增子中的应用。解链曲线分析是熟知的并在例如美国专利号6,174,670、6472156和6,569,627中描述，每一篇都通过引用的方式全文并入本文，特别是它们对于应用解链曲线分析以检测和/或定量扩增产物的描述。在示例的实施方案中，使用双链DNA染料，如SYBR Green、Pico Green(Molecular Probes,Inc.,Eugene,OR)、EVA Green(Biotinum)、溴化乙锭等等进行解链曲线分析(参见Zhu等人.,1994,Anal.Chem.66:1941-48)。By obtaining fluorescence at different temperatures, it is possible to track the degree of hybridization. In addition, the temperature dependence of PCR product hybridization can be used to identify and/or quantitative PCR products. Therefore, the method described herein includes the application of melting curve analysis in detection and/or quantitative amplicon. Melting curve analysis is well known and described in, for example, U.S. Patent Nos. 6,174,670, 6472156 and 6,569,627, each of which is incorporated herein by reference in its entirety, particularly for the application of melting curve analysis to detect and/or quantitative amplification product. In an illustrative embodiment, double-stranded DNA dyes such as SYBR Green, Pico Green (Molecular Probes, Inc., Eugene, OR), EVA Green (Biotinum), ethidium bromide, etc. are used to perform melting curve analysis (see Zhu et al., 1994, Anal. Chem. 66: 1941-48).

在某些实施方案中，多路检测在单独扩增混合物中进行，例如，在微流体装置的单独反应区室中，其可用于进一步增加在单个检验中可被分析的样品和/或靶的数目或用于进行比较性方法，诸如比较性基因组杂交(CGH)。在多个实施方案中，在每个单独反应区室中进行多达2、3、4、5、6、7、8、9、10、50、100、500、1000、5000、10000或更多个扩增反应。In certain embodiments, multiplexed detection is performed in separate amplification mixtures, e.g., in separate reaction compartments of a microfluidic device, which can be used to further increase the number of samples and/or targets that can be analyzed in a single test or for comparative methods, such as comparative genomic hybridization (CGH). In various embodiments, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000, 10000 or more amplification reactions are performed in each separate reaction compartment.

根据某些实施方案，技术人员可采用内部标准来定量由荧光信号所指示的扩增产物。参见，例如，美国专利号5,736,333。According to certain embodiments, one can employ an internal standard to quantify the amplification product indicated by the fluorescent signal. See, for example, U.S. Patent No. 5,736,333.

已经开发了可用包含荧光染料的组合物进行热循环反应、发射特定波长的光束、读取荧光染料的强度、并在每个循环后展示荧光强度的装置。包含热循环仪、光束发射器和荧光信号检测仪的装置已经描述在例如，美国专利号5,928,907；6,015,674；和6,174,670中。Devices have been developed that can perform thermal cycling reactions using compositions containing fluorescent dyes, emit a light beam of a specific wavelength, read the intensity of the fluorescent dye, and display the fluorescence intensity after each cycle. Devices comprising a thermal cycler, a light beam emitter, and a fluorescent signal detector are described, for example, in U.S. Patent Nos. 5,928,907; 6,015,674; and 6,174,670.

在一些实施方案中，这些功能的每一个可由分别的装置进行。例如，如果技术人员采用Q-beta复制酶反应用于扩增，反应可不在热循环仪中进行，但可包括在特定波长发射的光束，检测荧光信号，并计算和展示扩增产物的量。In some embodiments, each of these functions can be performed by separate devices. For example, if a technician uses a Q-beta replicase reaction for amplification, the reaction may not be performed in a thermal cycler, but may include emitting a light beam at a specific wavelength, detecting a fluorescent signal, and calculating and displaying the amount of amplified product.

在具体实施方案中，组合的热循环和荧光检测装置可用于准确定量靶核酸。在一些实施方案中，可在一个或多个热循环期间和/或之后检测和展示荧光信号，从而允许随着反应发生实时监测扩增产物。在某些实施方案中，技术人员可使用扩增产物的量和扩增循环的数目来计算扩增之前样品中存在多少靶核酸序列。In a specific embodiment, the thermal cycle and fluorescence detection device of the combination can be used for accurate quantitative target nucleic acid. In some embodiments, the fluorescent signal can be detected and displayed during and/or after one or more thermal cycles, thereby allowing real-time monitoring of the amplified product as the reaction occurs. In certain embodiments, the technician can use the amount of the amplified product and the number of amplification cycles to calculate how many target nucleic acid sequences are present in the sample before amplification.

用于DNA测序的扩增Amplification for DNA sequencing

在某些实施方案中，采用扩增方法以产生适于自动DNA测序的扩增子。许多现有的DNA测序技术依赖于"通过合成测序"。这些技术包括文库产生、文库分子的大型平行PCR扩增、和测序。文库产生开始于转变样品核酸为适当尺寸的片段、连接衔接子序列到片段末端、和选择适当地附带衔接子的分子。文库分子末端上衔接子序列的存在使得能够扩增随机-序列插入物。将核苷酸序列加标签的上述方法可代替连接以掺入衔接子序列，如以下更详细描述的。In certain embodiments, an amplification method is employed to generate amplicons suitable for automated DNA sequencing. Many existing DNA sequencing technologies rely on "sequencing by synthesis". These techniques include library generation, large-scale parallel PCR amplification of library molecules, and sequencing. Library generation begins with converting sample nucleic acids into fragments of appropriate size, ligating adapter sequences to the ends of the fragments, and selecting molecules that are appropriately accompanied by adapters. The presence of adapter sequences on the ends of the library molecules enables the amplification of random-sequence inserts. The above-described methods for tagging nucleotide sequences can be substituted for ligation to incorporate adapter sequences, as described in more detail below.

此外，上述方法提供靶核苷酸序列的大致上均匀的扩增的能力有助于制备具有良好覆盖的DNA测序文库。在自动DNA测序的上下文中，术语"覆盖"是指在测序时测量序列的次数。具有大致上均匀的覆盖的DNA测序文库可产生其中覆盖大致上也是均匀的序列数据。如此，在多个实施方案中，在进行如本文所述地制备的多个靶扩增子的自动测序时，靶扩增子的至少50％的序列以大于靶扩增子序列的平均拷贝数的50％和小于靶扩增子序列的平均拷贝数的2倍存在。在这一方法的多个实施方案中，靶扩增子序列的至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％以大于靶扩增子序列的平均拷贝数的50％和小于靶扩增子序列的平均拷贝数的2倍存在。In addition, the ability that the amplification that the above-mentioned method provides roughly uniformly of target nucleotide sequence helps to prepare the DNA sequencing library with good coverage.In the context of automatic DNA sequencing, term " coverage " refers to the number of times that sequence is measured when order-checking.The DNA sequencing library with roughly uniform coverage can produce sequence data that wherein coverage is also roughly uniform.So, in a plurality of embodiments, when carrying out the automatic sequencing of a plurality of target amplicons prepared as described herein, at least 50% of the sequence of target amplicons exists with greater than 50% of the average copy number of target amplicon sequence and less than 2 times of the average copy number of target amplicon sequence. In various embodiments of this method, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the target amplicon sequences are present at greater than 50% and less than 2 times the average copy number of the target amplicon sequences.

在某些实施方案中，可采用至少三个引物以产生适于DNA测序的扩增子：正向、反向和条形码引物。然而，正向引物、反向引物和条形码引物的一个或多个可包括至少一个另外的引物结合位点。在具体实施方案中，条形码引物包括至少第一另外的引物结合位点，其位于条形码核苷酸序列的上游，条形码核苷酸序列在第一核苷酸标签特异性部分的上游。在某些实施方案中，正向引物、反向引物和条形码引物的两个包括至少一个另外的引物结合位点(即，从而扩增产生的扩增子包括核苷酸标签序列、条形码核苷酸序列、和两个另外的结合位点)。例如，如果条形码引物包括条形码核苷酸序列上游的第一另外的引物结合位点，在具体实施方案中，反向引物可包括第二核苷酸标签下游的至少第二另外的引物结合位点。然后扩增产生具有以下元件的分子：5'-第一另外的引物结合位点-条形码核苷酸序列-来自正向引物的第一核苷酸标签-靶核苷酸序列-来自反向引物的第二核苷酸标签-第二另外的引物结合位点-3'。在具体实施方案中，第一和第二另外的引物结合位点能够被DNA测序引物结合，以帮助测序包含条形码的完整扩增子，如以上讨论的，条形码可指示样品来源。In certain embodiments, at least three primers may be employed to generate an amplicon suitable for DNA sequencing: a forward, a reverse, and a barcode primer. However, one or more of the forward primer, the reverse primer, and the barcode primer may include at least one additional primer binding site. In specific embodiments, the barcode primer includes at least a first additional primer binding site located upstream of the barcode nucleotide sequence, which is upstream of the first nucleotide tag-specific portion of the barcode nucleotide sequence. In certain embodiments, two of the forward primer, the reverse primer, and the barcode primer include at least one additional primer binding site (i.e., such that the amplicon generated by amplification includes the nucleotide tag sequence, the barcode nucleotide sequence, and two additional binding sites). For example, if the barcode primer includes a first additional primer binding site upstream of the barcode nucleotide sequence, in specific embodiments, the reverse primer may include at least a second additional primer binding site downstream of the second nucleotide tag. Amplification then generates a molecule having the following elements: 5' - first additional primer binding site - barcode nucleotide sequence - first nucleotide tag from the forward primer - target nucleotide sequence - second nucleotide tag from the reverse primer - second additional primer binding site - 3'. In specific embodiments, the first and second additional primer binding sites are capable of being bound by DNA sequencing primers to facilitate sequencing of the entire amplicon containing the barcode, which, as discussed above, can indicate the source of the sample.

在其他实施方案中，采用至少四个引物以产生适于DNA的扩增子。例如，内侧引物可与另外包含能够被DNA测序引物结合的第一和第二引物结合位点的外侧引物一起使用。扩增产生具有以下元件的分子：5'-第一引物结合位点-第二条形码核苷酸序列-第一核苷酸标签序列-第一条形码核苷酸序列-靶核苷酸序列-第一条形码核苷酸序列-第二核苷酸标签序列-第二条形码核苷酸序列-第二引物结合位点-3'。因为这一分子在任一末端包含条形码组合，可从分子的任一末端获得序列来辨识条形码组合。In other embodiments, at least four primers are used to generate an amplicon suitable for DNA. For example, an inner primer can be used with an outer primer that also contains first and second primer binding sites capable of being bound by DNA sequencing primers. Amplification produces a molecule having the following elements: 5'-first primer binding site-second barcode nucleotide sequence-first nucleotide tag sequence-first barcode nucleotide sequence-target nucleotide sequence-first barcode nucleotide sequence-second nucleotide tag sequence-second barcode nucleotide sequence-second primer binding site-3'. Because this molecule contains a barcode combination at either end, sequences can be obtained from either end of the molecule to identify the barcode combination.

以类似的方式，可采用六个引物来准备用于测序的DNA。更具体地，如以上讨论的内侧和填充引物，可与另外包含能够被DNA测序引物结合的第一和第二引物结合位点的外侧引物一起使用。扩增产生具有以下元件的分子：5'-第一引物结合位点-第二条形码核苷酸序列-第三核苷酸标签序列-第一条形码核苷酸序列-第一核苷酸标签序列-靶核苷酸序列-第二核苷酸标签序列-第一条形码核苷酸序列-第四核苷酸标签序列-第二条形码核苷酸序列-第二引物结合位点-3'。因为这一分子在任一末端包含条形码组合，可从分子的任一末端获得序列来辨识条形码组合。In a similar manner, six primers can be used to prepare DNA for sequencing. More specifically, the inner and filler primers discussed above can be used with outer primers that additionally contain first and second primer binding sites capable of being bound by DNA sequencing primers. Amplification produces a molecule having the following elements: 5'-first primer binding site-second barcode nucleotide sequence-third nucleotide tag sequence-first barcode nucleotide sequence-first nucleotide tag sequence-target nucleotide sequence-second nucleotide tag sequence-first barcode nucleotide sequence-fourth nucleotide tag sequence-second barcode nucleotide sequence-second primer binding site-3'. Because this molecule contains a barcode combination at either end, sequences can be obtained from either end of the molecule to identify the barcode combination.

本文描述的方法可包括利用任何可得的DNA测序方法对至少一个靶扩增子进行DNA测序。在具体实施方案中，利用高通量测序方法测序多个靶扩增子。此类方法通常利用体外克隆步骤来扩增单独的DNA分子。如以上讨论的，乳液PCR(emPCR)分离油相中的含水液滴中的单独DNA分子连同引物包被的珠。PCR产生DNA分子的拷贝，其结合珠上的引物，随后固定用于后面的测序。体外克隆扩增还可通过"桥式PCR"进行，其中引物附加到固体表面后扩增片段。物理结合到表面的DNA分子可平行地测序，例如，通过焦磷酸测序或通过如以上讨论的合成测序方法。Method described herein can comprise utilizing any available DNA sequencing method to carry out DNA sequencing to at least one target amplicon.In a specific embodiment, utilize high-throughput sequencing method to order-check a plurality of target amplicon.This type of method utilizes in vitro cloning step to increase independent DNA molecule conventionally.As discussed above, emulsion PCR (emPCR) separates the independent DNA molecule in the aqueous droplet in the oil phase together with the primer-coated pearl.PCR produces a copy of DNA molecule, and it is fixed for back order-checking subsequently in conjunction with the primer on the pearl.In vitro cloning amplification can also be carried out by " bridge PCR ", and wherein primer is attached to the amplification fragment after solid surface.The DNA molecule that is physically bound to surface can be ordered in parallel, for example, by pyrophosphate sequencing or by as above-discussed synthetic sequencing method.

标记策略Tagging strategy

可以将任何适合的标记策略用于本文所述的方法中。当测定混合物被等分，并且分析每个等份的单一扩增产物的存在时，可以将通用检测探针用于扩增混合物中。在具体实施方案中，可以使用通用qPCR探针来进行实时PCR检测。适当的通用qPCR探针包括双链DNA染料，例如SYBR Green、Pico Green(Molecular Probes,Inc.,Eugene,OR)、EVA Green(Biotinum)、溴化乙锭、以及类似物(参见Zhu等人,1994,Anal.Chem.66:1941-48)。适当的通用qPCR探针还包括序列特异性探针，这些探针结合到所有扩增产物中存在的核苷酸序列上。在扩增期间，这类探针的结合位点可以被方便地掺入到加标签的靶核苷酸序列中。Any suitable labeling strategy can be used in the methods described herein. When the assay mixture is divided equally and the presence of a single amplified product of each aliquot is analyzed, a universal detection probe can be used in the amplification mixture. In a specific embodiment, a universal qPCR probe can be used to perform real-time PCR detection. Suitable universal qPCR probes include double-stranded DNA dyes, such as SYBR Green, Pico Green (Molecular Probes, Inc., Eugene, OR), EVA Green (Biotinum), ethidium bromide, and analogs (see Zhu et al., 1994, Anal. Chem. 66: 1941-48). Suitable universal qPCR probes also include sequence-specific probes, which are bound to the nucleotide sequences present in all amplified products. During amplification, the binding site of such probes can be conveniently incorporated into the target nucleotide sequence of the label.

作为替代方案，可以将一个或多个靶特异性qPCR探针(即对于有待检测的靶核苷酸序列具有特异性的)用于扩增混合物中以对扩增产物进行检测。靶特异性探针可能是有用的，例如当在大量的样品中仅少量靶核酸有待检测时。例如，如果仅3个靶有待检测时，可以使用对于每个靶具有不同荧光标记物的靶特异性探针。通过正确地选择标记物，可以进行分析，其中在单一反应中不同标记物在不同波长下被激发和/或检测。参见，例如Fluorescence Spectroscopy(Pesce等人编著)Marcel Dekker,New York,(1971)；White等人,Fluorescence Analysis:A Practical Approach,Marcel Dekker,New York,(1970)；Berlman,Handbook of Fluorescence Spectra of Aromatic Molecules,第2版,AcademicPress,New York,(1971)；Griffiths,Colour and Constitution of Organic Molecules,Academic Press,New York,(1976)；Indicators(Bishop编著).Pergamon Press,Oxford,19723；和Haugland,Handbook of Fluorescent Probes and Research Chemicals,Molecular Probes,Eugene(1992)。Alternatively, one or more target-specific qPCR probes (i.e., specific for the target nucleotide sequence to be detected) can be used in the amplification mixture to detect the amplified product. Target-specific probes may be useful, for example, when only a small number of target nucleic acids are to be detected in a large sample. For example, if only three targets are to be detected, target-specific probes with different fluorescent labels for each target can be used. By properly selecting the labels, analyses can be performed in which different labels are excited and/or detected at different wavelengths in a single reaction. See, e.g., Fluorescence Spectroscopy (Pesce et al., eds.) Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Color and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, ed.). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992).

去除不想要的反应组分Removal of unwanted reaction components

应当理解的是其中使用多个反应步骤的涉及核酸的复杂混合物的反应可能导致多个未掺入的反应组分，并且通过多个提纯步骤(clean-up procedure)中任何一项去除这类未掺入的反应组分或降低它们的浓度可以提高随后发生反应的效率和特异性。例如，在一些实施方案中，在进行在此所述的扩增步骤之前，去除预扩增引物、或降低预扩增引物的浓度可能是令人希望的。It should be understood that reactions involving complex mixtures of nucleic acids using multiple reaction steps may result in a plurality of unincorporated reaction components, and that removing such unincorporated reaction components or reducing their concentration by any of a number of clean-up procedures may improve the efficiency and specificity of subsequent reactions. For example, in some embodiments, it may be desirable to remove preamplification primers, or reduce the concentration of preamplification primers, prior to performing the amplification steps described herein.

在某些实施方案中，可以通过简单地稀释来降低不希望的组分的浓度。例如，在进行扩增之前可以将预扩增的样品稀释约2倍、5倍、10倍、100倍、500倍、1000倍从而提高随后扩增步骤的特异性。In certain embodiments, the concentration of undesirable components can be reduced by simple dilution. For example, the pre-amplified sample can be diluted about 2-fold, 5-fold, 10-fold, 100-fold, 500-fold, 1000-fold before amplification to improve the specificity of the subsequent amplification step.

在一些实施方案中，可以通过多种酶学方法来去除不想要的组分。作为替代方案，或除了上述方法之外，可以通过纯化来去除不想要的组分。例如，可以将纯化标签掺入到上述引物的任何一个中(例如条形码核苷酸序列中)以有助于对加标签的靶核苷酸进行纯化。In some embodiments, unwanted components can be removed by various enzymatic methods. As an alternative, or in addition to the above methods, unwanted components can be removed by purification. For example, a purification tag can be incorporated into any of the above primers (e.g., a barcode nucleotide sequence) to facilitate purification of the tagged target nucleotide.

在具体实施方案中，提纯包括选择性地固定希望的核酸。例如，可以优先地将希望的核酸固定到固相支持体上。在示例性实施方案中，亲和性部分，例如生物素(例如，光-生物素)被附加到希望的核酸上，并且得到的生物素标记的核酸被固定在固体支持物上，该固体支持物包括亲和部分-结合物(affinity moiety-binder)，例如链霉抗生物素蛋白。可以用多个探针对固定的核酸进行查询，并且将未杂交的和/或未连接的探针通过洗涤去除(参见，例如，公布的P.C.T.申请WO 03/006677以及USSN 09/931,285)。作为替代方案，可以对固定的核酸进行洗涤以去除其他组分并且随后从该固体支持物中释放固定的核酸用于进一步分析。这种方法可以被用于例如在加入用于DNA测序的引物结合位点之后从扩增混合物中回收靶扩增子。在具体实施方案中，可以将亲和性部分例如生物素附加到扩增引物上这样使得扩增生成亲和性部分标记的(例如生物素标记的)扩增子。因此，例如当使用三个引物以将条形码和核苷酸标签元件加入到靶核苷酸序列中时，如上所述，条形码或反向引物中至少一个可以包括亲和性部分。当使用四个引物(两个内侧引物以及两个外侧引物)来将希望的元件加入到靶核苷酸序列中时，外侧引物中至少一个可以包括亲和性部分。In a specific embodiment, purification comprises the nucleic acid of selectively fixing hope.For example, the nucleic acid of hope can be preferentially fixed on solid support.In an exemplary embodiment, affinity moiety, for example biotin (for example, light-biotin) is attached on the nucleic acid of hope, and the nucleic acid of the biotin labeling obtained is fixed on solid support, and this solid support comprises affinity moiety-binding substance (affinity moiety-binder), for example streptavidin.Can inquire with multiple probes to fixing nucleic acid, and unhybridized and/or unconnected probe is removed by washing (referring to, for example, the P.C.T. application WO 03/006677 and USSN 09/931,285 of announcement).As an alternative, can wash to remove other components and discharge fixing nucleic acid subsequently from this solid support for further analysis to fixing nucleic acid.This method can be used to, for example, reclaim target amplicon from amplification mixture after adding the primer binding site for dna sequencing. In a specific embodiment, an affinity moiety, such as biotin, can be attached to the amplification primers so that amplification generates affinity moiety-labeled (e.g., biotin-labeled) amplicons. Thus, for example, when three primers are used to add a barcode and nucleotide tag elements to a target nucleotide sequence, as described above, at least one of the barcode or reverse primers can include an affinity moiety. When four primers (two inner primers and two outer primers) are used to add a desired element to a target nucleotide sequence, at least one of the outer primers can include an affinity moiety.

微流体装置Microfluidic devices

在某些实施方案中，可以使用微流体装置来实现本文所述的方法。在示例性实施方案中，该装置是矩阵型的微流体装置，允许同时将多个底物溶液(substrate solution)与多个试剂溶液组合在分别分离的反应区室(separate isolated reactioncompartment)中。应当理解的是底物溶液可以包含一个或多个底物(例如靶核酸)并且试剂溶液可以包含一个或多个试剂。例如，微流体装置可以允许同时配对地组合多个不同扩增引物和样品。在某些实施方案中，该装置被配置以在这些不同室的每一个中包含不同组合的引物和样品。在不同实施方案中，分离的反应区室的数量可以是大于50个、通常地大于100个、更经常地大于500个、甚至更经常地大于1000个、并且时常大于5000个、或大于10,000个。In certain embodiments, microfluidic device can be used to realize method described herein.In exemplary embodiments, this device is the microfluidic device of matrix type, allows multiple substrate solutions (substrate solution) and multiple reagent solutions to be combined in respectively separated reaction chamber (separate isolated reaction compartment) simultaneously.It should be understood that substrate solution can comprise one or more substrates (such as target nucleic acid) and reagent solution can comprise one or more reagents.For example, microfluidic device can allow to combine multiple different amplification primers and sample in pairs simultaneously.In certain embodiments, this device is configured to comprise primer and sample of different combinations in each of these different chambers.In different embodiments, the quantity of the reaction chamber of separation can be greater than 50, generally greater than 100, more often greater than 500, even more often greater than 1000 and often greater than 5000 or greater than 10,000.

在具体实施方案中，矩阵型的微流体装置是Dynamic Array("DA")微流体装置。DA微流体装置是被设计用于分离配对组合的样品和试剂(例如，扩增引物、检测探针、等)并且适合用于进行定性以及定量PCR反应包括实时定量PCR分析的矩阵型微流体装置。在一些实施方案中，这种DA微流体装置至少部分是由弹性体制造的。DA微流体装置被描述于PCT公布号WO05107938 A2(Thermal Reaction Device and Method For Using The Same)和美国专利公布号US20050252773A1中，出于它们的DA微流体装置的说明的目的，这两者都通过引用以其全部内容并入本文。DA微流体装置可以并入多个高密度矩阵设计，这些设计使用该微流体装置的层之间的流体连通通路来编织穿过该装置并且在层之间的控制管线以及流体管线。通过弹性体块的多个层中的流体管线，高密度反应单元(reaction cell)安排是可能的。作为替代方案，可以设计DA微流体装置使得所有试剂以及样品通道都位于同一弹性体层中，控制通道在不同的层中。在某些实施方案中，DA微流体装置可用于将M个数目的不同的样品与N个数目的不同的试剂反应。In a specific embodiment, the matrix-type microfluidic device is a Dynamic Array ("DA") microfluidic device. The DA microfluidic device is a matrix-type microfluidic device designed for separating paired combinations of samples and reagents (e.g., amplification primers, detection probes, etc.) and suitable for performing qualitative and quantitative PCR reactions, including real-time quantitative PCR analysis. In some embodiments, such a DA microfluidic device is at least partially made of an elastomer. The DA microfluidic device is described in PCT Publication No. WO05107938 A2 (Thermal Reaction Device and Method For Using The Same) and U.S. Patent Publication No. US20050252773A1, both of which are incorporated herein by reference in their entirety for the purpose of their description of the DA microfluidic device. The DA microfluidic device can incorporate multiple high-density matrix designs that use fluid communication pathways between the layers of the microfluidic device to weave control lines and fluid lines through the device and between the layers. High-density reaction cell arrangements are possible through the fluid lines in multiple layers of an elastomer block. Alternatively, the DA microfluidic device can be designed so that all reagents and sample channels are located in the same elastomer layer, with the control channels in a different layer. In certain embodiments, the DA microfluidic device can be used to react M number of different samples with N number of different reagents.

虽然WO05107938中所述的DA微流体装置非常适合用于进行在本文所述的方法，本发明不限于任何具体的装置或设计。可以使用分配样品和/或允许独立的配对组合试剂和样品的任何装置。美国专利公布号20080108063(该专利通过引用以其全部内容并入本文)包括说明了48.48DYNAMIC ARRAY^TM IFC，可以从Fluidigm Corp.(South San FranciscoCalif)得到的一种可商购的装置的图表。应当理解的是其他构型是可能的并且是被考虑的，例如48×96、96×96、30×120；等。While the DA microfluidic device described in WO05107938 is well suited for carrying out the methods described herein, the present invention is not limited to any particular device or design. Any device that distributes the sample and/or allows for independent pairing and combining of reagents and samples may be used. U.S. Patent Publication No. 20080108063 (which is incorporated herein by reference in its entirety) includes a diagram illustrating a 48.48 DYNAMIC ARRAY^™ IFC, a commercially available device available from Fluidigm Corp. (South San Francisco, Calif.). It should be understood that other configurations are possible and are contemplated, such as 48×96, 96×96, 30×120; etc.

在具体实施方案中，微流体装置可以是DIGITAL ARRAY^TM IFC微流体装置，该装置适于进行数字扩增。这类装置可以具有将样品和试剂的混合物分配到纳升体积反应区室中的整合的通道以及阀。在一些实施方案中，DIGITAL ARRAY^TM IFC微流体装置至少部分是由弹性体制造的。示例性的DIGITAL ARRAY^TM IFC微流体装置被描述于共同未决的属于Fluidigm Corp.(South San Francisco,CA)的美国申请中，例如标题为“Method andApparatus for Determining Copy Number Variation Using Digital PCR”的美国申请号12/170,414。一个示例性实施方案具有12个输入端口，这些端口相应于12个单独的样品输入该装置中。该装置可以具有12个嵌板(panel)，并且这12个嵌板的每一个可以包含765个6 nL的反应区室，每个嵌板总体积为4.59μL。微流体通道可以将嵌板上的不同的反应区室连接到流体来源上。可以将压力施加到一个累加器(accumulator)以开放和关闭将反应区室连接到流体来源上的阀。在示例性实施方案中，可以提供12个入口用于载入样品试剂混合物。可以使用48个入口来提供试剂来源，当将压力施加到累加器上时，试剂被供应到芯片上。另外地，可以提供两个或更多个入口以提供芯片的水合。In specific embodiments, the microfluidic device can be a DIGITAL ARRAY^™ IFC microfluidic device, which is suitable for performing digital amplification. Such devices can have integrated channels and valves for dispensing a mixture of sample and reagents into nanoliter-volume reaction chambers. In some embodiments, the DIGITAL ARRAY^™ IFC microfluidic device is fabricated at least in part from an elastomer. Exemplary DIGITAL ARRAY^™ IFC microfluidic devices are described in co-pending U.S. applications to Fluidigm Corp. (South San Francisco, CA), such as U.S. application Ser. No. 12/170,414, entitled “Method and Apparatus for Determining Copy Number Variation Using Digital PCR.” One exemplary embodiment has 12 input ports, corresponding to 12 separate sample inputs into the device. The device can have 12 panels, and each of the 12 panels can contain 765 6 nL reaction chambers, for a total volume of 4.59 μL per panel. The microfluidic channel can connect the different reaction compartments on the panel to the fluid source. Pressure can be applied to an accumulator (accumulator) to open and close the valve that the reaction compartment is connected to the fluid source. In an exemplary embodiment, 12 inlets can be provided for loading sample reagent mixture. 48 inlets can be used to provide a reagent source, and when pressure is applied to the accumulator, reagent is supplied to the chip. Additionally, two or more inlets can be provided to provide hydration of the chip.

尽管DIGITAL ARRAY^TM IFC微流体装置非常适合用于实现在本文所述的某些扩增方法，本领域普通技术人员应当认识到对于这些装置的多种变更和替代方案。给定的DIGITAL ARRAY^TM IFC微流体装置的几何形状将取决于具体应用。与适合用于在本文所述方法中的装置相关的另外的说明被提供于美国专利申请公布号20050252773中，出于它的披露DIGITAL ARRAY^TM IFC微流体装置的目的，通过引用将其并入本文。Although DIGITAL ARRAY^™ IFC microfluidic devices are well suited for implementing certain amplification methods described herein, those skilled in the art will recognize a variety of variations and alternatives to these devices. The geometry of a given DIGITAL ARRAY^™ IFC microfluidic device will depend on the specific application. Additional description of devices suitable for use in the methods described herein is provided in U.S. Patent Application Publication No. 20050252773, which is incorporated herein by reference for its disclosure of DIGITAL ARRAY^™ IFC microfluidic devices.

在某些实施方案中，可以使用提供反应产物的回收的微流体装置来实现在本文所述的方法。这类装置被详细描述于2009年4月2日提交的共同未决的美国申请号61/166,105中(该专利通过引用以其全部内容并入本文并且特别地是出于它的说明允许反应产物回收的微流体装置以及相关方法的目的)，并由Fluidigm Corp.作为ACCESS ARRAY^TM IFC(Integrated Fluidic Circuit)出售。In certain embodiments, the methods described herein can be implemented using a microfluidic device that provides for recovery of the reaction products. Such a device is described in detail in co-pending U.S. application Ser. No. 61/166,105, filed Apr. 2, 2009 (which is hereby incorporated by reference in its entirety and particularly for its description of a microfluidic device that allows for recovery of the reaction products and related methods), and is sold by Fluidigm Corp. as the ACCESS ARRAY^™ IFC (Integrated Fluidic Circuit).

在这一类型的示例性装置中，独立的样品输入与引物输入以MxN阵列构型组合。因此，每个反应是特定样品与特定试剂混合物的独特组合。在一个实现方式中，通过安排为列的样品输入管线将样品载入微流体装置的样品区室中。通过安排为横过所述列的行的测定液输入管线将测定试剂(例如引物)载入微流体装置的测定区室中。在加载期间样品区室和测定区室是处于流体隔绝的。在加载过程完成之后，将可操作以防止流体管线通过样品和测定区室的对之间的接口阀开放以使样品和测定液的配对组合能够自由地界面间扩散。样品和测定液的精确混合使不同配对组合之间能够发生反应，在每个区室中生成一个或多个反应产物。收获反应产物并且然后可将其用于随后的过程。如在此使用的术语“测定液(assay)”和“样品”是这些装置在某些实施方案中的具体用途的说明。然而，这些装置的用途不限于在所有实施方案中使用样品”以及“测定液”。例如，在其他实施方案中，“样品”可以指“第一试剂”或多个“第一试剂”并且“测定液”可以指“第二试剂”或多个“第二试剂”。这些装置的MxN特征使有待组合的第一试剂的任何组能够与第二试剂的任何组相组合。In this type of exemplary device, independent sample input and primer input are combined with MxN array configuration. Therefore, each reaction is a unique combination of a specific sample and a specific reagent mixture. In one implementation, the sample is loaded into the sample compartment of the microfluidic device by the sample input line arranged in a row. The assay reagent (such as a primer) is loaded into the assay compartment of the microfluidic device by the assay liquid input line arranged in the row across the row. During loading, the sample compartment and the assay compartment are in fluid isolation. After the loading process is completed, it will be operable to prevent the fluid line from being opened by the interface valve between the pair of sample and assay compartment so that the paired combination of sample and assay liquid can freely diffuse between the interfaces. The precise mixing of sample and assay liquid enables reaction to occur between different paired combinations, generating one or more reaction products in each compartment. The harvest reaction product and then can be used for subsequent processes. The terms "assay liquid (assay)" and "sample" as used herein are the descriptions of the specific uses of these devices in certain embodiments. However, the use of these devices is not limited to the use of "sample" and "assay fluid" in all embodiments. For example, in other embodiments, "sample" can refer to a "first reagent" or multiple "first reagents" and "assay fluid" can refer to a "second reagent" or multiple "second reagents." The MxN feature of these devices enables any set of first reagents to be combined with any set of second reagents.

根据具体实施方案，可以将来自MxN个配对组合的反应产物从微流体装置回收到离散的池中，例如，M个样品的每一个对应一个池。典型地，离散的池被包含在提供于载体(carrier)上的样品输入端口中。在一些方法中，出于归一化的目的，可以在“每个扩增子”的基础上收集这些反应产物。使用本发明的实施方案，有可能实现其中扩增产物的拷贝数在一个样品中改变不大于±25％并且在样品间不大于±25％的结果(针对从样品和测定液的同一输入溶液组装的重复实验)。因此，如通过特定已知基因型分布测量的，从微流体装置中回收的扩增产物将是输入样品的代表。在某些实施方案中，输出样品浓度将大于2,000个拷贝/扩增子/微升，并且反应产物的回收将在小于两小时内完成。According to specific embodiments, the reaction product from MxN paired combinations can be recovered in discrete pools from microfluidic devices, for example, each corresponding pool of M samples. Typically, discrete pools are included in the sample input port provided on a carrier. In some methods, for normalized purposes, these reaction products can be collected on the basis of "each amplicon". Using embodiments of the present invention, it is possible to achieve that the copy number of amplified product changes no more than ± 25% in a sample and no more than ± 25% result between samples (for repeated experiments assembled from the same input solution of sample and assay liquid). Therefore, as measured by specific known genotype distribution, the amplified product recovered from microfluidic devices will be the representative of the input sample. In certain embodiments, the output sample concentration will be greater than 2,000 copies/amplicons/microliters, and the recovery of reaction product will be completed in less than two hours.

在一些实施方案中，反应产物由膨胀泵送回收。膨胀泵送提供了使用常规技术通常不可获得的益处。例如，膨胀泵送使得能够从微流体装置缓慢去除反应产物。在示例的实施方案中，以小于100μl/小时的流体流速回收反应产物。在这个示例中，为了将48个反应产物分配到每一列中的反应区室，每个反应产物的体积为约1.5μl，在约30分钟的时间段中去除反应产物将导致72μl/小时的流体流速。(即，48x1.5/0.5小时)。在其他实施方案中，反应产物的去除速率以以下速率进行：小于90μl/hr、80μl/hr、70μl/hr、60μl/hr、50μl/hr、40μl/hr、30μl/hr、20μl/hr、10μl/hr、9μl/hr、小于8μl/hr、小于7μl/hr、小于6μl/hr、小于5μl/hr、小于4μl/hr、小于3μl/hr、小于2μl/hr、小于1μl/hr或小于0.5μl/hr。In some embodiments, the reaction products are recovered by expansion pumping. Expansion pumping provides benefits that are generally not available using conventional techniques. For example, expansion pumping enables the slow removal of reaction products from the microfluidic device. In an exemplary embodiment, the reaction products are recovered at a fluid flow rate of less than 100 μl/hour. In this example, in order to distribute 48 reaction products to the reaction chambers in each column, the volume of each reaction product is approximately 1.5 μl, and removing the reaction products over a period of approximately 30 minutes will result in a fluid flow rate of 72 μl/hour. (i.e., 48 x 1.5/0.5 hours). In other embodiments, the removal rate of the reaction products is less than 90 μl/hr, 80 μl/hr, 70 μl/hr, 60 μl/hr, 50 μl/hr, 40 μl/hr, 30 μl/hr, 20 μl/hr, 10 μl/hr, 9 μl/hr, less than 8 μl/hr, less than 7 μl/hr, less than 6 μl/hr, less than 5 μl/hr, less than 4 μl/hr, less than 3 μl/hr, less than 2 μl/hr, less than 1 μl/hr or less than 0.5 μl/hr.

膨胀泵送导致清除微流体装置中存在的大致上高百分比的以及可能地所有的反应产物。一些实施方案去除微流体装置的反应区室(例如，样品区室)中存在的多于75％的反应产物。作为举例，一些实施方案去除反应区室中存在的反应产物的多于80％、85％、90％、92％、95％、96％、97％、98％或99％。Expansion pumping results in the removal of a substantially high percentage, and potentially all, of the reaction products present in the microfluidic device. Some embodiments remove more than 75% of the reaction products present in a reaction chamber (e.g., a sample chamber) of the microfluidic device. By way of example, some embodiments remove more than 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the reaction products present in the reaction chamber.

本文描述的方法可使用带有多个一般包括样品区室和检验区室的"单元小室"的微流体装置。此类单元小室可具有数百微米数量级尺寸，例如具有如下尺寸的单元小室：500x500μm、525x525μm、550x550μm、575x575μm、600x600μm、625x625μm、650x650μm、675x675μm、700x700μm或类似尺寸。选择样品区室和测定区室的尺寸以提供当减少样品和测定液用量时足以完成希望过程的材料量。作为举例，样品区室可以具有100-400μm宽度x200-600μm长度x100-500μm高度数量级的尺寸。例如，宽度可以是100μm、125μm、150μm、175μm、200μm、225μm、250μm、275μm、300μm、325μm、350μm、375μm、400μm或类似尺寸。例如，长度可以是200μm、225μm、250μm、275μm、300μm、325μm、350μm、375μm、400μm、425μm、450μm、475μm、500μm、525μm、550μm、575μm、600μm或类似尺寸。例如，高度可以是100μm、125μm、150μm、175μm、200μm、225μm、250μm、275μm、300μm、325μm、350μm、375μm、400μm、425μm、450μm、475μm、500μm、525μm、550μm、575μm、600μm或类似尺寸。测定区室可以具有类似尺寸范围，这典型地提供了比较小的区室体积更小的范围内类似的步长(step size)。在一些实施方案中，样品区室体积对于测定区室体积的比率是约5:1、10:1、15:1、20:1、25:1或30:1。比列出的范围更小的区室体积被包括在本发明的范围内并且易于使用微流体装置制造技术来制造。The methods described herein can utilize a microfluidic device with multiple "unit cells," typically comprising a sample compartment and an assay compartment. Such unit cells can have dimensions on the order of hundreds of micrometers, for example, unit cells having dimensions of 500 x 500 μm, 525 x 525 μm, 550 x 550 μm, 575 x 575 μm, 600 x 600 μm, 625 x 625 μm, 650 x 650 μm, 675 x 675 μm, 700 x 700 μm, or similar dimensions. The dimensions of the sample compartment and assay compartment are selected to provide a sufficient amount of material to complete the desired process while minimizing sample and assay fluid usage. By way of example, a sample compartment can have dimensions on the order of 100-400 μm in width, 200-600 μm in length, and 100-500 μm in height. For example, the width can be 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, or the like. For example, the length can be 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm, 450 μm, 475 μm, 500 μm, 525 μm, 550 μm, 575 μm, 600 μm, or the like. For example, the height can be 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 325 μm, 350 μm, 375 μm, 400 μm, 425 μm, 450 μm, 475 μm, 500 μm, 525 μm, 550 μm, 575 μm, 600 μm or similar size. The assay chamber can have a similar size range, which typically provides a similar step size in a smaller range than a smaller compartment volume. In some embodiments, the sample compartment volume is about 5:1, 10:1, 15:1, 20:1, 25:1 or 30:1 for the ratio of the assay compartment volume. Compartment volumes smaller than the listed range are included within the scope of the present invention and are easy to manufacture using microfluidic device manufacturing technology.

更高密度的微流体装置典型地将使用更小的区室体积以减少单元小室的覆盖区(footprint)。在其中可以得到非常小样品尺寸的应用中，减少区室体积将有助于对此类小的样品进行测试。Higher density microfluidic devices will typically use smaller compartment volumes to reduce the footprint of the unit cell. In applications where very small sample sizes are available, reducing compartment volumes will facilitate testing of such small samples.

对于单颗粒分析，可设计微流体装置以帮助上样和捕获待分析的特定颗粒。图9显示用于分析哺乳动物细胞的示例性的微流体装置的单元小室结构。每个单元小室具有"细胞通道"(即，样品区室)和"检验通道"(即，检验区室)。细胞通道是圆形的，用于上样哺乳动物细胞，尺寸为直径在数十微米级别至长度在一百数百微米。取决于被分析的细胞的尺寸，直径可以是约15μm、约20μm、约25um、约30μm、约35μm、约40μm、或约45μm或更多，或可落入以这些值的任一个作为端点的范围内。取决于被分析的细胞的尺寸，长度可以是约60μm、约90μm、约120μm、约150μm、约170μm、约200μm、约230μm、约260μm、约290μm或更多，或可落入以这些值的任一个作为端点的范围内。在基于ACCESS ARRAY^TM IFC平台("MA006")的示例性的微流体装置中，用于上样哺乳动物细胞的单元小室可以是约30μmx170μm。这样的装置可配置为在上样后对细胞通道提供或帮助提供热来溶解细胞。如图9所示，装置可包括与细胞通道分开的检验通道用于进行反应诸如核酸扩增。170μmx170封锁阀(containment valve)可用于关闭细胞通道。For single particle analysis, microfluidic devices can be designed to help sample loading and capture specific particles to be analyzed. Figure 9 shows the unit cell structure of an exemplary microfluidic device for analyzing mammalian cells. Each unit cell has a "cell channel" (i.e., a sample compartment) and a "test channel" (i.e., a test compartment). The cell channel is circular and is used to load mammalian cells, with a size ranging from a diameter of tens of microns to a length of hundreds of microns. Depending on the size of the cell being analyzed, the diameter can be about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, or about 45 μm or more, or can fall within the range of any one of these values as an endpoint. Depending on the size of the cell being analyzed, the length can be about 60 μm, about 90 μm, about 120 μm, about 150 μm, about 170 μm, about 200 μm, about 230 μm, about 260 μm, about 290 μm or more, or can fall within the range of any one of these values as an endpoint. In an exemplary microfluidic device based on the ACCESS ARRAY^™ IFC platform ("MA006"), the unit cell for loading mammalian cells can be approximately 30 μm x 170 μm. Such a device can be configured to provide or assist in providing heat to the cell channel after loading to lyse the cells. As shown in Figure 9, the device can include a separate test channel from the cell channel for performing reactions such as nucleic acid amplification. A 170 μm x 170 containment valve can be used to close the cell channel.

2012年2月29日提交的题为"Methods,Systems,And Devices For MultipleSingle-Particle or Single-Cell Processing Using Microfluidics"的共同待决的美国申请号61/605,016描述了用于利用微流体的多个单颗粒或单细胞处理的方法、系统和装置。多个实施方案提供从细胞颗粒的较大群体捕获、分隔、和/或操作单独颗粒或细胞，以及产生与每个单独颗粒或细胞相关的遗传信息和/或反应。一些实施方案可配置为成像单独颗粒或细胞或相关的反应产物作为处理的部分。这一申请通过引用全文并入本文，尤其是其对配置用于多个单颗粒或单细胞处理的微流体装置和相关系统的描述。Co-pending U.S. application No. 61/605,016, filed February 29, 2012, and entitled “Methods, Systems, and Devices for Multiple Single-Particle or Single-Cell Processing Using Microfluidics,” describes methods, systems, and devices for multiple single-particle or single-cell processing using microfluidics. Various embodiments provide for capturing, isolating, and/or manipulating individual particles or cells from a larger population of cell particles, as well as generating genetic information and/or reactions associated with each individual particle or cell. Some embodiments may be configured to image individual particles or cells or associated reaction products as part of the processing. This application is hereby incorporated by reference in its entirety, particularly for its description of microfluidic devices and related systems configured for multiple single-particle or single-cell processing.

在具体实施方案中，采用微流体装置以有助于具有下面的动态范围的测定法：至少3个数量级、更经常是至少4个、至少5个、至少6个、至少7个或至少8个数量级。In specific embodiments, microfluidic devices are employed to facilitate assays having a dynamic range of at least 3 orders of magnitude, more often at least 4, at least 5, at least 6, at least 7, or at least 8 orders of magnitude.

使用弹性体材料的制造方法以及用于设计装置以及它们的部件的方法已经详细地描述于科学以及专利文献中。参见，例如，Unger等人.(2000)Science 288:113-116；美国专利号US 6,960,437(Nucleic acid amplification utilizing microfluidicdevices)；6,899,137(Microfabricated elastomeric valve and pump systems)；6,767,706(Integrated active flux microfluidic devices and methods)；6,752,922(Microfluidic chromatography)；6,408,878(Microfabricated elastomeric valve andpump systems)；6,645,432(microfluidic devices including three-dimensionallyarrayed channel networks)；美国专利申请公布号2004/0115838；2005/0072946；2005/0000900；2002/0127736；2002/0109114；2004/0115838；2003/0138829；2002/0164816；2002/0127736；和2002/0109114；PCT公布号WO2005/084191；WO 05/030822A2；和WO 01/01025；Quake&Scherer,2000,"From micro to nanofabrication with soft materials"Science 290:1536-40；Unger等人.,2000,"Monolithic microfabricated valves andpumps by multilayer soft lithography"Science 288:113-116；Thorsen等人.,2002,"Microfluidic large-scale integration"Science 298:580-584；Chou等人.,2000,"Microfabricated Rotary Pump"Biomedical Microdevices 3:323-330；Liu等人.,2003,"Solving the"world-to-chip"interface problem with a microfluidic matrix"Analytical Chemistry 75,4718-23,Hong等人,2004,"A nanoliter-scale nucleic acidprocessor with parallel architecture"Nature Biotechnology22:435-39。Fabrication methods using elastomeric materials and methods for designing devices and their components have been described in detail in the scientific and patent literature. See, for example, Unger et al. (2000) Science 288:113-116; U.S. Patent Nos. 6,960,437 (Nucleic acid amplification utilizing microfluidic devices); 6,899,137 (Microfabricated elastomeric valve and pump systems); 6,767,706 (Integrated active flux microfluidic devices and methods); 6,752,922 (Microfluidic chromatography); 6,408,878 (Microfabricated elastomeric valve and pump systems); 6,645,432 (microfluidic devices including three-dimensionally arrayed channel networks); U.S. Patent Application Publication Nos. 2004/0115838; 2005/0072946; 2005/0000900; 2002/0127736; 2002/0109114; 2004/0115838; 2003/0138829; 2002/0164816; 2002/0127736; and 2002/0109114; PCT Publication Nos. WO 2005/084191; WO 05/030822 A2; and WO 01/01025; Quake & Scherer, 2000, "From micro to nanofabrication with soft materials," Science 290:1536-40; Unger et al., 2000, "Monolithic microfabricated valves and pumps by multilayer soft lithography" Science 288:113-116; Thorsen et al., 2002, "Microfluidic large-scale integration" Science 298:580-584; Chou et al., 2000, "Microfabricated Rotary Pump" Biomedical Microdevices 3:323-330; Liu et al., 2003, "Solving the "world-to-chip" interface problem with a microfluidic matrix" Analytical Chemistry 75, 4718-23, Hong et al., 2004, "A nanoliter-scale nucleic acid processor with parallel architecture" Nature Biotechnology 22: 435-39.

数据输出和分析Data output and analysis

在某些实施方案中，当在矩阵型的微流体装置上执行本文所述的方法时数据能够作为热矩阵(也称为“热图”)输出。在热矩阵中，每个正方形(代表DA矩阵上的反应区室)已经被指定颜色值，该颜色值能够以灰度形式显示，但更典型地以颜色显示。在灰度中，黑色正方形指示未检测出扩增产物，而白色正方形指示扩增产生的最高水平，其中灰度阴影指示其间扩增产物的水平。在进一步方面中，可以使用软件程序来对将热矩阵中生成的数据编译成读者更友好的形式。In certain embodiments, when the methods described herein are performed on a matrix-type microfluidic device, the data can be output as a thermal matrix (also referred to as a "heat map"). In the thermal matrix, each square (representing a reaction compartment on the DA matrix) has been assigned a color value, which can be displayed in grayscale, but is more typically displayed in color. In the grayscale, black squares indicate that no amplification product was detected, while white squares indicate the highest level of amplification, with grayscale shading indicating the level of amplification product in between. In a further aspect, a software program can be used to compile the data generated in the thermal matrix into a more reader-friendly format.

应用application

在具体实施方案中，本文所述的方法用于分析一个或多个核酸，例如(在一些实施方案中)，颗粒中或与颗粒缔合的一个或多个核酸。因此，例如，这些方法适用于鉴别特定多态性(例如SNP)、等位基因、或单体型、或染色体异常例如扩增、缺失、重排、或非整倍性的存在。这些方法可以被用于基因分型中，基因分型可以在多种背景中实现，这些背景包括对遗传疾病或疾患、癌症进行诊断、药物基因组学(个体化用药)、农业中质量控制(例如用于种子或家畜)、对植物或动物的种群进行研究和管理(例如，水产养殖或渔业管理中或确定种群多样性中)、或亲子或法医鉴定。本文所述的方法可以用于在生物或环境样品中鉴别指示特定条件或生物体的序列。例如，所述方法可以用于测定中以鉴别病原体例如，病毒、细菌、以及真菌。所述方法还可以被用于旨在表征环境或微环境的研究中，例如表征人肠中的微生物物种。In a specific embodiment, the methods described herein are used to analyze one or more nucleic acids, such as (in some embodiments), in a particle or with one or more nucleic acids associated with a particle. Therefore, for example, these methods are applicable to differentiating specific polymorphisms (such as SNPs), alleles or haplotypes or chromosomal abnormalities such as amplification, deletion, rearrangement or the presence of aneuploidy. These methods can be used in genotyping, and genotyping can be realized in a variety of backgrounds, including diagnosing genetic diseases or illnesses, cancer, pharmacogenomics (individualized medication), quality control in agriculture (such as for seeds or livestock), studying and managing plant or animal populations (such as, in aquaculture or fishery management or determining population diversity), or parentage or forensic identification. The methods described herein can be used to identify the sequence of an indication specific condition or organism in biological or environmental samples. For example, the method can be used in determining to identify pathogens such as, viruses, bacteria, and fungi. The method can also be used to characterize the research of environment or microenvironment, such as characterizing the microbial species in human intestine.

在某些实施方案中，这些方法还可以用于确定DNA或RNA拷贝数。确定基因组DNA中异常DNA拷贝数是有用的，例如，在诊断和/或预后遗传缺陷和疾病，例如癌中。对于感兴趣的基因，例如在不同条件下(例如，不同外部刺激或疾病状态)和/或在不同发育阶段下，例如不同个体、组织、或细胞中的表达监控而言，确定RNA“拷贝数”，即表达水平，是有用的。In certain embodiments, these methods can also be used to determine DNA or RNA copy number. Determining that abnormal DNA copy number in genomic DNA is useful, for example, in diagnosis and/or prognosis of genetic defects and diseases, such as cancer. For gene of interest, for example, under different conditions (for example, different external stimuli or disease states) and/or at different developmental stages, for example, for expression monitoring in different individuals, tissues or cells, determining RNA " copy number", i.e. expression level, is useful.

此外，可以使用所述方法来制备核酸样品用于进一步分析，例如像DNA测序。Furthermore, the methods can be used to prepare nucleic acid samples for further analysis, such as, for example, DNA sequencing.

而且，在随后分析之前，作为第一步骤，可以将核酸样品加标签，从而降低样品的错误标记或交叉污染将损害结果的风险。例如，在收集之后任何医师办公室、实验室、或医院可以立即对样品进行加标签，并且在分析时可以对标签进行确认。类似地，可以对包含在犯罪现场收集的核酸的样品尽快加标签，以确保样品不被错误标记或被篡改。样品从一方到另一方的每次传递时的标签检测可以用于建立样品的保管链。Moreover, before subsequent analysis, as a first step, nucleic acid samples can be labeled, thereby reducing the risk that the mislabeling or cross contamination of the sample will damage the result. For example, any physician's office, laboratory, or hospital can label the sample immediately after collection, and can confirm the label when analyzing. Similarly, the sample of the nucleic acid collected at the crime scene can be labeled as soon as possible to ensure that the sample is not mislabeled or tampered with. Label detection during each transfer of the sample from one party to the other can be used to set up the chain of custody of the sample.

如以上讨论的，除了核酸以外，本文描述的方法还可用于分析颗粒的其他参数，诸如例如，每个颗粒中或与每个颗粒缔合的一个或多个蛋白的表达水平。在一些实施方案中，对每个颗粒分析一个或多个核酸连同一个或多个其他参数。As discussed above, in addition to nucleic acids, the methods described herein can also be used to analyze other parameters of the particles, such as, for example, the expression level of one or more proteins in or associated with each particle. In some embodiments, one or more nucleic acids are analyzed for each particle along with one or more other parameters.

将多个参数的检验结果与颗粒群体中每个颗粒相关联的能力可在多种不同类型研究中开发。在多个实施方案中，本文描述的方法可用于鉴定两种或更多种变化诸如拷贝数变化、突变、表达水平变化或剪接变体，其中将变化一起与表型关联。表型可以是，例如，疾病的风险、存在、严重度、预后、和/或对具体疗法的响应性或对药物的耐药性。本文描述的方法还可用于检测特定核酸序列的共存在，其可指示基因组重组、特定剪接变体的共表达、B细胞中特定轻链和重链的共表达。该方法还可适用于检测特定宿主细胞中特定病原体的存在，例如，当病原体特异性和宿主细胞特异性核酸二者(或其他参数)共同存在于相同的细胞中时。该方法还可用于从循环中的肿瘤细胞靶向再测序，例如，在不同癌症中的突变热点。The ability to associate the test results of multiple parameters with each particle in a particle population can be developed in a variety of different types of studies. In various embodiments, the methods described herein can be used to identify two or more changes such as copy number changes, mutations, expression level changes, or splice variants, wherein the changes are associated with a phenotype. A phenotype can be, for example, the risk, presence, severity, prognosis, and/or responsiveness to a specific therapy or resistance to a drug for a disease. The methods described herein can also be used to detect the co-existence of specific nucleic acid sequences, which can indicate genomic recombination, co-expression of specific splice variants, co-expression of specific light and heavy chains in B cells. The method can also be applied to detecting the presence of specific pathogens in specific host cells, for example, when both pathogen-specific and host cell-specific nucleic acids (or other parameters) are present in the same cell. The method can also be used for targeted resequencing from circulating tumor cells, for example, mutation hotspots in different cancers.

试剂盒Reagent test kit

根据本发明的试剂盒可包括对于实践本文描述的一个或多个测定方法有用的一个或多个试剂。总体上试剂盒包括包装，其中一个或多个容器容纳试剂(例如引物和/或探针)作为一个或多个单独组合物，或任选地，当试剂的相容性将允许时，作为混合物。该试剂盒还可以包括从使用者的观点来看可以是令人希望的其他物质，例如缓冲剂、稀释剂、标准品、和/或在样品处理、洗涤、或进行该测定的任何其他步骤中有用的任何其他物质。在具体实施方案中，试剂盒包括以上讨论的一个或多个矩阵型微流体装置。Test kit according to the present invention may include one or more reagents useful for practicing one or more assay methods described herein. Generally, test kit includes packaging, wherein one or more containers hold reagents (such as primers and/or probes) as one or more individual compositions, or optionally, when the compatibility of reagent will allow, as a mixture. The test kit may also include other substances that may be desirable from the user's point of view, such as buffers, diluents, standards, and/or any other substances useful in sample treatment, washing, or any other step of carrying out the assay. In a specific embodiment, the test kit includes one or more matrix-type microfluidic devices discussed above.

在某些实施方案中，本发明包括用于进行加入衔接子分子到包含粘末端的多个靶核酸的每个末端的上述方法的试剂盒。这些实施方案可用于，例如，用于高通量DNA测序的片段产生。这样的试剂盒可包含设计为用于这一方法的多个衔接子分子(参见以上)和选自以下组成的组的一个或多个组分：DNA酶、核酸外切酶、核酸内切酶、聚合酶和连接酶。。In certain embodiments, the present invention includes kits for performing the above-described methods of adding adapter molecules to each end of a plurality of target nucleic acids comprising sticky ends. These embodiments can be used, for example, for fragment generation for high-throughput DNA sequencing. Such kits can include a plurality of adapter molecules designed for use in this method (see above) and one or more components selected from the group consisting of: a DNA enzyme, an exonuclease, an endonuclease, a polymerase, and a ligase.

在具体实施方案中，本发明包括用于组合条形码化的试剂盒。用于进行四引物方法的试剂盒，例如，可包含聚合酶和：In a specific embodiment, the present invention includes a kit for combinatorial barcoding. A kit for performing a four-primer method, for example, can include a polymerase and:

(i)内侧引物，包含：(i) an inner primer comprising:

正向、内侧引物，包含第一核苷酸标签、第一条形码核苷酸序列和靶特异性部分；和a forward, inner primer comprising a first nucleotide tag, a first barcode nucleotide sequence, and a target-specific portion; and

反向、内侧引物，包含靶特异性部分、第一条形码核苷酸序列和第二核苷酸标签；和a reverse, inner primer comprising a target-specific portion, a first barcode nucleotide sequence, and a second nucleotide tag; and

(ii)外侧引物，包含：(ii) outer primers, comprising:

正向、外侧引物，包含第二条形码核苷酸序列和第一核苷酸标签特异性部分；和a forward, outer primer comprising a second barcode nucleotide sequence and a first nucleotide tag-specific portion; and

反向、外侧引物，包含第二核苷酸标签特异性部分和第二条形码核苷酸序列，其中外侧引物是比内侧引物过量的。用于进行六引物、组合条形码化方法的试剂盒可包含聚合酶和：A reverse, outer primer comprising a second nucleotide tag-specific portion and a second barcode nucleotide sequence, wherein the outer primer is in excess relative to the inner primer. A kit for performing the six-primer, combinatorial barcoding method may comprise a polymerase and:

(i)内侧引物，包含：(i) an inner primer comprising:

正向、内侧引物，包含第一核苷酸标签和靶特异性部分；和a forward, inner primer comprising a first nucleotide tag and a target-specific portion; and

反向、内侧引物，包含靶特异性部分和第二核苷酸标签；a reverse, inner primer containing a target-specific portion and a second nucleotide tag;

(ii)填充引物，包含：(ii) a filler primer comprising:

正向、填充引物，包含第三核苷酸标签、第一条形码核苷酸序列和第一核苷酸标签特异性部分；和a forward, filler primer comprising a third nucleotide tag, a first barcode nucleotide sequence, and a portion specific for the first nucleotide tag; and

反向、填充引物，包含第二核苷酸标签特异性部分、第一条形码核苷酸序列、第四核苷酸标签；和a reverse, filler primer comprising a second nucleotide tag-specific portion, a first barcode nucleotide sequence, a fourth nucleotide tag; and

(iii)外侧引物，包含：(iii) outer primers, comprising:

正向、外侧引物，包含第二条形码核苷酸序列和第三核苷酸标签特异性部分；和a forward, outer primer comprising a second barcode nucleotide sequence and a third nucleotide tag-specific portion; and

反向、外侧引物，包含第四核苷酸标签特异性部分和第二条形码核苷酸序列；其中外侧引物是比填充引物过量的，填充引物是比内侧引物过量的。A reverse, outer primer comprises a fourth nucleotide tag-specific portion and a second barcode nucleotide sequence; wherein the outer primer is present in excess relative to the stuffer primer, which is present in excess relative to the inner primer.

在其他实施方案中，本发明包括用于基于组合连接的加标签的试剂盒。这些试剂盒包含多个衔接子，包含：In other embodiments, the present invention includes kits for combinatorial ligation-based tagging. These kits contain a plurality of adaptors, including:

多个第一衔接子，各自包含相同的核酸内切酶位点、N个不同的条形码核苷酸序列、第一引物结合位点和粘末端，其中N是大于1的整数；a plurality of first adaptors, each comprising an identical endonuclease site, N different barcode nucleotide sequences, a first primer binding site, and sticky ends, wherein N is an integer greater than 1;

第二衔接子，包含第二引物结合位点和粘末端；和a second adaptor comprising a second primer binding site and sticky ends; and

多个第三衔接子，包含第二条形码核苷酸序列和与在所述核酸内切酶位点切割所述第一衔接子时产生的那些互补的粘末端，其中多个第三衔接子包含M个不同的第二条形码核苷酸序列，其中M是大于1的整数。这样的试剂盒可任选地包含对第一衔接子中的核酸内切酶位点特异性的核酸内切酶和/或连接酶。a plurality of third adaptors comprising a second barcode nucleotide sequence and sticky ends complementary to those produced upon cleavage of the first adaptor at the endonuclease site, wherein the plurality of third adaptors comprises M different second barcode nucleotide sequences, where M is an integer greater than 1. Such a kit may optionally comprise an endonuclease and/or a ligase specific for the endonuclease site in the first adaptor.

本发明还提供用于通过插入诱变来加标签的试剂盒，其也可用于如上所述的组合加标签。在某些实施方案中，这样的试剂盒包含：The present invention also provides a kit for tagging by insertional mutagenesis, which can also be used for combinatorial tagging as described above. In certain embodiments, such a kit comprises:

一个或多个核苷酸标签；和one or more nucleotide tags; and

多个条形码引物，其中每个条形码引物包含：A plurality of barcoded primers, wherein each barcoded primer comprises:

第一部分，其是对核苷酸标签的第一部分特异性的，连接于；a first portion, which is specific for the first portion of the nucleotide tag, attached to;

条形码核苷酸序列，其不退火到核苷酸标签，连接于；a barcode nucleotide sequence, which does not anneal to a nucleotide tag, is attached;

第二部分，其是对核苷酸标签的第二部分特异性的，其中多个条形码引物各自包含相同的第一和第二标签特异性部分、但M个不同的第二条形码核苷酸序列，其中M是大于1的整数。在具体实施方案中，核苷酸标签包含转座子末端，且试剂盒另外包含转座酶，转座酶可将转座子末端加入到靶核酸。这样的试剂盒还可任选包含聚合酶。A second portion that is specific for the second portion of the nucleotide tag, wherein the plurality of barcode primers each comprise the same first and second tag-specific portions but M different second barcode nucleotide sequences, where M is an integer greater than 1. In a specific embodiment, the nucleotide tag comprises a transposon end, and the kit further comprises a transposase that can add the transposon end to the target nucleic acid. Such a kit may also optionally comprise a polymerase.

本发明包括可用于双向核酸测序的试剂盒。在具体实施方案中，这样的试剂盒可包含：The present invention includes a kit that can be used for bidirectional nucleic acid sequencing. In a specific embodiment, such a kit may include:

第一外侧引物组，其中该组包含：A first outer primer set, wherein the set comprises:

第一外侧、正向引物，包含对第一引物结合位点特异性的部分；和a first outer, forward primer comprising a portion specific for the first primer binding site; and

第一外侧、反向引物，包含条形码核苷酸序列和对第二引物结合位点特异性的部分，其中第一和第二引物结合位点是不同的；a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for a second primer binding site, wherein the first and second primer binding sites are different;

第二外侧引物组，其中该组包含：a second outer primer set, wherein the set comprises:

第二外侧、正向引物，包含条形码核苷酸序列和对第一引物结合位点特异性的部分；和a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and

第二外侧、反向引物，包含对第二引物结合位点特异性的部分。在某些实施方案中，第一和第二引物结合位点可以是DNA测序引物的结合位点。在一些实施方案中，外侧引物可各自另外包含另外的核苷酸序列，其中：The second outer, reverse primer comprises a portion specific for the second primer binding site. In certain embodiments, the first and second primer binding sites can be binding sites for DNA sequencing primers. In some embodiments, the outer primers can each additionally comprise an additional nucleotide sequence, wherein:

第一外侧、正向引物包含第一另外的核苷酸序列，且第一外侧、反向引物包含第二另外的核苷酸序列；和The first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and

第二外侧、正向引物包含第二另外的核苷酸序列，且第二外侧、反向引物包含第一另外的核苷酸序列；且第一和第二另外的核苷酸序列是不同的。在具体、示例性实施方案中，第一外侧引物组包含PE1-CS1和PE2-BC-CS2，且第二外侧引物组包含PE1-CS2和PE2-BC-CS1(表1，实施例9)。The second outer, forward primer comprises a second additional nucleotide sequence, and the second outer, reverse primer comprises a first additional nucleotide sequence; and the first and second additional nucleotide sequences are different. In a specific, exemplary embodiment, the first outer primer set comprises PE1-CS1 and PE2-BC-CS2, and the second outer primer set comprises PE1-CS2 and PE2-BC-CS1 (Table 1, Example 9).

包含两组外侧引物的双向核酸测序试剂盒还可任选地包含内侧引物组，其中该组包含：A bidirectional nucleic acid sequencing kit comprising two sets of outer primers may optionally further comprise an inner primer set, wherein the set comprises:

内侧、正向引物，包含靶特异性部分和第一引物结合位点；和an inner, forward primer comprising a target-specific portion and a first primer binding site; and

内侧、反向引物，包含靶特异性部分和第二引物结合位点。在某些实施方案中，试剂盒可包含各自对不同的靶核酸特异性的多个内侧引物组。An inner, reverse primer comprising a target-specific portion and a second primer binding site. In certain embodiments, a kit may comprise multiple sets of inner primers, each specific for a different target nucleic acid.

这些双向核酸测序试剂盒的任一个还可任选地包含以下DNA测序引物：Any of these bidirectional nucleic acid sequencing kits may also optionally contain the following DNA sequencing primers:

结合第一和第二引物结合位点并引发靶核苷酸序列的测序；和/或binds to the first and second primer binding sites and initiates sequencing of the target nucleotide sequence; and/or

结合第一和第二引物结合位点并引发条形码核苷酸序列的测序。在具体实施方案中，两个类型的DNA测序引物被包括在试剂盒中，且结合第一和第二引物结合位点并引发条形码核苷酸序列的测序的引物是引发靶核苷酸序列的测序的引物的反向互补物。在具体、示例性实施方案中，试剂盒包含DNA测序引物CS1、CS2、CS1rc和CS2rc(表2，实施例9)。Binds to the first and second primer binding sites and initiates sequencing of the barcode nucleotide sequence. In a specific embodiment, two types of DNA sequencing primers are included in the kit, and the primers that bind to the first and second primer binding sites and initiate sequencing of the barcode nucleotide sequence are the reverse complements of the primers that initiate sequencing of the target nucleotide sequence. In a specific, exemplary embodiment, the kit comprises DNA sequencing primers CS1, CS2, CS1rc, and CS2rc (Table 2, Example 9).

试剂盒通常上包括用于实现本文所述的一种或多种方法的说明书。试剂盒中包括的说明书可以被附着在包装材料上或可以作为包装插入物(package insert)而包括。虽然这些说明书典型地是书面或打印的材料，它们不限于此。能够存储这类说明书并且使它们与最终用户进行交流的任何介质是本发明考虑的。这类介质包括但不限于电子存储介质(例如，磁盘、磁带、盒带、芯片)、光学介质(例如CD ROM)、RF标签，等。如在此使用的，术语“说明书”可以包括提供该说明书的互联网的地址。The kit typically includes instructions for implementing one or more methods described herein. The instructions included in the kit can be attached to the packaging material or can be included as a package insert. Although these instructions are typically written or printed materials, they are not limited to this. Any medium capable of storing such instructions and communicating them with the end user is contemplated by the present invention. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, cassettes, chips), optical media (e.g., CD ROMs), RF tags, etc. As used herein, the term "instructions" may include the address of an Internet site that provides the instructions.

应当理解，本文所述的实例及实施方案仅是为说明目的且并且根据其的不同的修饰或变化将对本领域中的普通技术人员进行建议，并将包括于本申请的精神及范围以及随附权利要求的范围内。It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or variations thereof will be suggested to those skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

此外，出于所有的目的，在此引用的所有其他出版物、专利、以及专利申请都通过引用以其全部内容并入本文。Furthermore, all other publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

实施例Example

实施例1Example 1

用于DNA测序的一般文库制备方法General library preparation methods for DNA sequencing

制备用于核酸测序的文库的现有方法是繁琐的，需要多个步骤。方法的关键包括随机片段化DNA(例如)，随后末端修复，抛光片段末端和连接末端衔接子。这些步骤各需要特定反应条件和在每个步骤之间纯化产物。Existing methods for preparing libraries for nucleic acid sequencing are cumbersome and require multiple steps. Key components of the method include random fragmentation of DNA (for example), followed by end-repair, polishing of the fragment ends, and ligation of end adapters. Each of these steps requires specific reaction conditions and product purification between each step.

这一实施例与图1和2描述了文库制备的可选方法。这一方法利用简并测序衔接子，其可以是双链DNA分子，包含用于给定测序仪的末端衔接子(或其部分)、限制性酶消化位点(或其他特异性裂解位点)、和在两条链3'末端的侧翼简并序列。可选地，衔接子可以是发夹序列或双链寡核苷酸。末端衔接子是在3'末端带有简并序列的单链寡核苷酸也是可能的。This example and Figures 1 and 2 describe an alternative method for library preparation. This method utilizes degenerate sequencing adapters, which can be double-stranded DNA molecules containing end adapters (or portions thereof) for a given sequencer, restriction enzyme digestion sites (or other specific cleavage sites), and flanking degenerate sequences at the 3' ends of both chains. Alternatively, the adapters can be hairpin sequences or double-stranded oligonucleotides. It is also possible that the end adapters are single-stranded oligonucleotides with degenerate sequences at the 3' end.

DNA将利用标准方法(例如酶促消化、喷雾化、超声处理)片段化。酶促消化将是优选的，因为它们对DNA分子用于下游步骤导致较少损伤。例如，可将DNA酶I加入待测序的DNA。这一反应可通过热处理终止。DNA will be fragmented using standard methods (e.g., enzymatic digestion, atomization, ultrasonication). Enzymatic digestion will be preferred because it causes less damage to the DNA molecules for downstream steps. For example, DNase I can be added to the DNA to be sequenced. This reaction can be terminated by heat treatment.

然后将在NTP不存在下利用T4聚合酶或无聚合酶活性的链特异性核酸外切酶在末端消化双链DNA回到单链DNA。核酸外切酶将是优选的，因为它可在单个反应中与连接酶(例如，热稳定连接酶)和聚合酶(例如，)一起作用。然而，如果使用T4聚合酶，制备方法仍将以多个步骤作用。The double-stranded DNA will then be digested back to single-stranded DNA at the end using T4 polymerase or a strand-specific exonuclease without polymerase activity in the absence of NTPs. Exonucleases are preferred because they can act together with a ligase (e.g., a thermostable ligase) and a polymerase (e.g., ) in a single reaction. However, if T4 polymerase is used, the preparation method will still act in multiple steps.

核酸酶消化将在DNA的末端暴露一条链。衔接子序列将在聚合酶和连接酶存在下加入。衔接子序列将退火到消化的DNA，缺口将被聚合酶/连接酶混合物填充和修复。在这一方案的一个版本中，衔接子序列将从发夹结构制造，从而在消化/连接/聚合期间，终产物是环化的DNA。其将被保护不受核酸外切酶的进一步降解，导致终产物的聚积。Nuclease digestion will expose one strand at the end of the DNA. Adapter sequences will be added in the presence of polymerase and ligase. The adapter sequences will anneal to the digested DNA, and the gaps will be filled and repaired by the polymerase/ligase mixture. In one version of this approach, the adapter sequences will be made from hairpin structures, so that during the digestion/ligation/polymerization process, the end product is circularized DNA. This will be protected from further degradation by exonucleases, resulting in the accumulation of the end product.

实施例2Example 2

用于Illumina测序的基于组合连接的条形码化Combinatorial ligation-based barcoding for Illumina sequencing

制备DNA测序文库，且标准PE2-BC-标签序列被RE-1-BC-标签代替。DNA sequencing libraries were prepared and the standard PE2-BC-tag sequence was replaced by the RE-1-BC-tag.

条形码序列的PE2标签序列下游用限制性酶(例如BsrD1)的识别位点(RE-1)代替，留下短的悬垂物：The PE2 tag sequence downstream of the barcode sequence is replaced with a recognition site (RE-1) for a restriction enzyme (e.g., BsrD1), leaving a short overhang:

5’-TGCATAGCAATGNN|CTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-靶-3’(SEQ IDNO：2)5’-TGCATAGCAATGNN|CTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-target-3’ (SEQ IDNO: 2)

3’-ACGTATCGTTAC|NNGATCCACTGACCTCAAGTCTGCACACGAGAAGGCTAGA-靶-5’(SEQ IDNO：3)3’-ACGTATCGTTAC|NNGATCCACTGACCTCAAGTCTGCACACGAGAAGGCTAGA-target-5’ (SEQ IDNO: 3)

用酶切割文库。The library is cleaved with an enzyme.

连接包含适当悬垂物的衔接子分子和第二条形码序列：Ligate adapter molecules containing appropriate overhangs and the second barcode sequence:

5'-CAAGCAGAAGACGGCATACGAGATAGCTNN+CTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-靶-3’(SEQ ID N0：4)5'-CAAGCAGAAGACGGCATACGAGATAGCTNN+CTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-target-3' (SEQ ID NO: 4)

3’-GTTCGTCTTCTGCCGTATGCTCTATCGA NNGATCCACTGACCTCAAGTCTGCACACGAGAAGGCTAGA-靶-5’(SEQ ID N0：5)3’-GTTCGTCTTCTGCCGTATGCTCTATCGA NNGATCCACTGACCTCAAGTCTGCACACGAGAAGGCTAGA-target-5’ (SEQ ID NO: 5)

连接将产生以下构建体：The ligation will produce the following construct:

5'-CAAGCAGAAGACGGCATACGAGATAGCTNNCTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-靶-3'(SEQ ID N0：6)5'-CAAGCAGAAGACGGCATACGAGATAGCTNNCTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-target-3' (SEQ ID NO: 6)

3'-GTTCGTCTTCTGCCGTATGCTCTATCGANNGATCCACTGACCTCAAGTCTGCACACGAGAAGGCTAGA-靶-5'(SEQ ID NO：7)3'-GTTCGTCTTCTGCCGTATGCTCTATCGANNGATCCACTGACCTCAAGTCTGCACACGAGAAGGCTAGA-target-5' (SEQ ID NO: 7)

利用标准清除方法在测序前去除剩余的衔接子分子。Standard cleanup methods were used to remove remaining adapter molecules prior to sequencing.

在测序运行上的索引读取期间，报告回的索引序列将是：CTAGNNAGCT(SEQ ID NO:8)。During the index read on a sequencing run, the index sequence reported back would be: CTAGNNAGCT (SEQ ID NO: 8).

实施例3Example 3

基因表达的单细胞分析Single-cell analysis of gene expression

问题：为了利用DYNAMIC ARRAY^TM IFC获得一组基因的单细胞基因表达数据，首先在芯片外的试管中分离细胞。分离这一细胞的方法是难以进行的和/或需要大量细胞。当细胞有限时，诸如来自组织的原代细胞和/或来自微孔板中的药物筛选试验的细胞，这一最后的障碍更多地变成利用BioMark从单细胞获得基因表达数据的屏障。Problem : To obtain single-cell gene expression data for a panel of genes using DYNAMIC ARRAY^™ IFC, cells must first be isolated in a test tube outside the chip. Methods for isolating these cells can be difficult and/or require large numbers of cells. When cells are limited, such as primary cells from tissues and/or cells from drug screening assays in microplates, this final hurdle becomes even more of a barrier to obtaining gene expression data from single cells using BioMark.

解决方案：ACCESS ARRAY^TM IFC("芯片")或允许回收反应混合物的类似芯片可用于经由有限稀释上样单细胞(例如MA006芯片)。通过利用芯片作为分选和制备用于下游基因表达分析的细胞的设备，可方便地为DYNAMIC ARRAY^TM IFC准备有限数目的细胞，从而为以上列出的问题提供解决方案。本发明的步骤如下：Solution : The ACCESS ARRAY^™ IFC ("chip") or similar chips that allow for the recovery of reaction mixtures can be used to load single cells via limiting dilution (e.g., the MA006 chip). By utilizing the chip as a device for sorting and preparing cells for downstream gene expression analysis, a limited number of cells can be conveniently prepared for DYNAMIC ARRAY^™ IFC, thereby providing a solution to the problems listed above. The steps of the present invention are as follows:

1)以有限稀释在ACCESS ARRAY^TM IFC中上样细胞。如图7A所示地上样引物组。任何给定的细胞将被暴露于所有基因特异性引物和单个独特条形码引物。1) Load cells at limiting dilution in the ACCESS ARRAY^™ IFC. Load primer sets as shown in Figure 7A. Any given cell will be exposed to all gene-specific primers and a single unique barcode primer.

2)在芯片中进行逆转录和预扩增。产生的扩增子的一个实例显示在图7B。这是3引物方法。使用这一方法的益处是，对特定实验仅需要设计和订购一组96个引物对(或更多，对于期望的许多基因)。BC反向引物是通用的，用在所有实验中。任何给定细胞将具有扩增的所有基因，且所有扩增子将已经被单个条形码加标签。(参见以下可能的变化)。2) Reverse transcription and pre-amplification are performed in the chip. An example of the resulting amplicon is shown in Figure 7B. This is a 3-primer approach. The benefit of using this approach is that only a set of 96 primer pairs (or more, for as many genes as desired) needs to be designed and ordered for a specific experiment. The BC reverse primer is universal and used in all experiments. Any given cell will have all genes amplified, and all amplicons will have been tagged with a single barcode. (See possible variations below).

3)反应产物由池(与不同的引物组成90度，即由样品)输出。池N现在包含96个基因(或更多或更少)的预扩增物与条形码的混合物，其中一个条形码与一个细胞匹配。池保持分离，从而即使多个细胞以相同的条形码加标签，它们也是可区分的，因为它们属于不同的池。3) The reaction products are pooled (with different primers at 90°, i.e., by sample). Pool N now contains a mixture of preamplified products for 96 genes (or more or less) and barcodes, where one barcode matches a cell. The pools remain separate, so even if multiple cells are tagged with the same barcode, they are distinguishable because they belong to different pools.

4)上样DYNAMIC ARRAY^TM IFC，如图7C所示。注意：单细胞可经由多种方法在ACCESSARRAY^TM芯片上追踪。这提供关于哪个池和哪个条形码预扩增反应具有单细胞的信息，即，哪个应被上样到DYNAMIC ARRAY^TM IFC上。这一选择允许我们仅读取包含一个细胞的ACCESSARRAY^TM IFC室，导致DYNAMIC ARRAY^TM IFC的有效使用。而且，如果目标细胞通过使用细胞特异性染色剂即对细胞表面标志物的抗体被圈定，则仅这一亚组的细胞可被选择用于上样到DYNAMIC ARRAY^TM IFC。当细胞在细胞的混杂群体中是罕见的即，干细胞、癌症干细胞、癌细胞的时候，这可变得重要。4) Loading DYNAMIC ARRAY^™ IFC, as shown in Figure 7C. Note: Single cells can be tracked on the ACCESSARRAY^™ chip via a variety of methods. This provides information about which pool and which barcoded pre-amplification reaction contains a single cell, i.e., which should be loaded onto the DYNAMIC ARRAY^™ IFC. This selection allows us to read only the ACCESSARRAY^™ IFC chamber containing a single cell, leading to efficient use of the DYNAMIC ARRAY^™ IFC. Furthermore, if the target cells are circled using cell-specific stains, i.e., antibodies to cell surface markers, only this subset of cells can be selected for loading onto the DYNAMIC ARRAY^™ IFC. This can be important when the cells are rare in a mixed population of cells, i.e., stem cells, cancer stem cells, cancer cells.

5)运行qPCR，用EvaGreen用于检测。通过扩增一个BC引物和一个基因特异性引物的组合，可获得单细胞(其扩增子在ACCESS ARRAY^TM IFC中的预扩增期间被BC引物加标签)对给定基因(其扩增将被DYNAMIC ARRAY^TM IFC中的基因特异性引物检测)的基因表达。5) Run qPCR using EvaGreen for detection. By amplifying a combination of a BC primer and a gene-specific primer, gene expression of a given gene (whose amplicon will be detected by the gene-specific primer in the DYNAMIC ARRAY^™ IFC) in a single cell (whose amplicon is tagged by the BC primer during pre-amplification in the ACCESS ARRAY^™ IFC) can be obtained.

可能的变化：存在具有预扩增一组基因和用独特条形码加标签单独细胞的共同最终结果的不同检测方法。实例如下：Possible variations : There are different detection methods with the common end result of pre-amplifying a panel of genes and labeling individual cells with unique barcodes. Examples are as follows:

如以上相同地进行但使用2-引物方法。Proceed identically as above but using the 2-primer method.

使用Fen-连接酶链式反应。Fen-ligase chain reaction was used.

使用解链温度策略。Use a melting temperature strategy.

实施例4Example 4

检测来自实施例3的反应产物的可选方法Alternative Method for Detecting Reaction Products from Example 3

代替利用qPCR以EvaGreen检测来自在ACCESS ARRAY^TM IFC中预扩增的BC-加标签的扩增子，在DYNAMIC ARRAY^TM IFC(例如，M96)中进行带有实时检测的连接酶链式反应。Instead of using qPCR with EvaGreen to detect BC-tagged amplicons from pre-amplification in an ACCESS ARRAY^™ IFC, ligase chain reaction with real-time detection was performed in a DYNAMIC ARRAY^™ IFC (eg, M96).

示例性的扩增子具有以下结构：5'-正向引物序列-靶核苷酸序列-反向引物序列-条形码核苷酸序列-3'。在这种情形中，一个引物可退火到反向引物序列，另一个引物可退火到邻近的条形码核苷酸序列，随后是连接、和重复的退火和连接循环。参见图8A。具有任一不同的反向引物("R")的池中的扩增子来源于不同的靶核酸(此处是信使RNA)，具有不同的条形码引物("BC")的池中的扩增子将不被扩增。因此，用BC_M扩增池_N扩增了来自ACCESSARRAY^TM IFC的行N、列M中的室的条形码化的靶核酸。在这一扩增中使用R₁作为另一个引物扩增了来源于对应R₁的靶核酸的扩增子。An exemplary amplicon has the following structure: 5'-forward primer sequence-target nucleotide sequence-reverse primer sequence-barcode nucleotide sequence-3'. In this case, one primer can anneal to the reverse primer sequence, and the other primer can anneal to the adjacent barcode nucleotide sequence, followed by connection, and repeated annealing and connection cycles. See Figure 8A. Amplicons in pools with any different reverse primers ("R") are derived from different target nucleic acids (here, messenger RNA), and amplicons in pools with different barcode primers ("BC") will not be amplified. Therefore, the barcoded target nucleic acid from the chamber in row N and column M of ACCESSARRAY^™ IFC was amplified using BC_M amplification pool_N. In this amplification, R₁ was used as another primer to amplify the amplicon derived from the target nucleic acid corresponding to R₁ .

实时检测的一个方法是悬垂片核酸内切酶-连接酶链式反应，其使用5'悬垂片核酸内切酶和标记的BC_n引物，如图8B所示。这一反应采用标记的探针和未标记的探针，其中探针对反应产物的同时杂交导致在标记的探针的5'末端形成悬垂片，且悬垂片的裂解可裂解荧光团与猝灭剂分开，产生信号。由于BC不是扩增子特异性的，这些引物仅需制造一次。例如，96个BC的一组对于任何数目的不同组F_nR_n个扩增子将是足够的。One method for real-time detection is the overhang sheet endonuclease-ligase chain reaction, which uses a 5' overhang sheet endonuclease and labeled BC_n primers, as shown in Figure 8 B. This reaction uses a labeled probe and an unlabeled probe, wherein the simultaneous hybridization of the probe pair reaction product causes the formation of an overhang sheet at the 5' end of the labeled probe, and the cracking of the overhang sheet can separate the cleavable fluorophore from the quencher to produce a signal. Since BC is not amplicon specific, these primers only need to be manufactured once. For example, a group of 96 BCs will be sufficient for any number of different groups of F_n R_n amplicons.

这一策略的益处：Benefits of this strategy:

池和BC的选择允许分析仅包含单细胞的那些ACCESS ARRAY^TM IFC室(其中单细胞分析是目的)。未标记的细胞可利用ACCESS ARRAY^TM IFC的亮视野或荧光成像来检测。此外，细胞可在上样到ACCESS ARRAY^TMIFC之前或之后用染料和/或标记的抗体染色以辨识感兴趣的细胞(例如，干细胞、癌细胞、癌症干细胞等等)。池和BC的选择允许分析仅包含感兴趣的细胞的那些ACCESS ARRAY^TM IFC室，改进效率。The selection of pools and BCs allows analysis of only those ACCESS ARRAY^™ IFC chambers containing single cells (where single cell analysis is the goal). Unlabeled cells can be detected using bright field or fluorescence imaging of ACCESS ARRAY^™ IFC. In addition, cells can be stained with dyes and/or labeled antibodies before or after loading onto ACCESS ARRAY^™ IFC to identify cells of interest (e.g., stem cells, cancer cells, cancer stem cells, etc.). The selection of pools and BCs allows analysis of only those ACCESS ARRAY^™ IFC chambers containing cells of interest, improving efficiency.

这一策略需要比FACS少的多的细胞，这使得可能用在不能利用FACS进行的分析中，诸如分析原代细胞或来自筛选试验的细胞的群体。This strategy requires far fewer cells than FACS, making it possible to use in analyses that cannot be performed using FACS, such as analysis of primary cells or populations of cells from screening assays.

实施例5Example 5

利用适于细胞操作的ACCESS ARRAY^TM IFC("MA006")从单细胞制备用于测序的核Preparation of nuclei for sequencing from single cells using ACCESS ARRAY^™ IFC ("MA006") for cell manipulation酸的方法Acid Method

一般方法的概述Overview of the general approach

"芯片"在本文称为MA006，已经利用ACCESS ARRAY^TM IFC平台开发，还开发了利用MA006、整合细胞操作和用于核酸测序的样品制备的方法。参见图9的MA006单元小室结构的示意图，显示芯片上方法。这一整合简化了执行实验所需的步骤。而且，上样芯片需要仅数百细胞。The "chip," referred to herein as MA006, has been developed using the ACCESS ARRAY^™ IFC platform. Methods have also been developed that utilize MA006 to integrate cell manipulation and sample preparation for nucleic acid sequencing. See Figure 9 for a schematic diagram of the MA006 unit cell architecture, illustrating the on-chip approach. This integration simplifies the steps required to perform an experiment. Furthermore, loading the chip requires only a few hundred cells.

MA006芯片具有以下特征：The MA006 chip has the following features:

单元小室具有170x30pm圆形通道来上样哺乳动物细胞The unit chamber has a 170x30pm circular channel for loading mammalian cells

48.48矩阵格式；48.48 matrix format;

在细胞通道中利用热溶解细胞；Using heat to lyse cells in the cell channel;

用于扩增反应的分别的反应区室；separate reaction compartments for amplification reactions;

170x170pm封锁阀以关闭细胞通道；170x170pm blocking valve to close cell channel;

额外的阻力层：PourOB-30gm圆形阻力；Additional resistance layers: PourOB - 30gm round resistance;

芯片制造：使用现有的AA48.48方法；Chip manufacturing: Use the existing AA48.48 method;

65pm准直容差；65pm collimation tolerance;

130pm冲孔直径；130pm punching diameter;

65x85pm阀尺寸；和65x85pm valve size; and

3层设计方法。3-tier design approach.

MA006芯片上没有细胞捕获特征。结果是有限稀释策略用于获得每室期望数目的细胞。然而，细胞捕获特征可被设计到芯片中。它们可以是物理(例如，杯或盏结构)、生物(例如，布点的肽)、或化学的(例如，带电荷离子)。The MA006 chip does not have cell-capturing features. Consequently, a limiting dilution strategy is used to obtain the desired number of cells per chamber. However, cell-capturing features can be designed into the chip. These can be physical (e.g., cup or calvaria structures), biological (e.g., dotted peptides), or chemical (e.g., charged ions).

芯片外的细胞操作：将待分析的细胞准备到使得获得每样品室(图9中的"细胞通道")期望数目的细胞的密度。因为MA006芯片使用有限稀释策略，每室细胞的数目在理论和实际上都遵循泊松分布。在第一种情况中，因为包含单细胞的最大数目的室是期望的，最佳细胞密度是每微升300-600细胞。1至2微升的最小体积可被施加到入口。因此，实验可用仅数百细胞进行。可使用来自任何来源(即，活生物体、组织培养物等等)的任何细胞类型(即，哺乳动物、细菌等等)。可使用制备、洗涤、和/或染色的任何形式或程度，只要其与下游应用相容。Cell manipulation outside the chip: The cells to be analyzed are prepared to a density that allows the desired number of cells to be obtained per sample chamber ("cell channel" in Figure 9). Because the MA006 chip uses a limiting dilution strategy, the number of cells per chamber follows a Poisson distribution in theory and practice. In the first case, because the chamber containing the maximum number of single cells is desired, the optimal cell density is 300-600 cells per microliter. A minimum volume of 1 to 2 microliters can be applied to the inlet. Therefore, the experiment can be carried out with only hundreds of cells. Any cell type (i.e., mammals, bacteria, etc.) from any source (i.e., living organisms, tissue cultures, etc.) can be used. Any form or degree of preparation, washing, and/or staining can be used, as long as it is compatible with downstream applications.

芯片中的细胞追踪：在任何聚合酶/扩增依赖性化学反应不存在下，可利用亮视野或荧光显微术监测芯片中细胞的位置、身份、和/或内容物。细胞可用任何染色剂染色(即，核酸特异性染色，诸如SYT010；免疫检测，诸如Cy5共轭的抗CD19；等等)，只要其与下游应用相容。这可用于例如在混杂的细胞群体中鉴定罕见细胞，即，癌症干细胞。Cell tracking within the chip: In the absence of any polymerase/amplification-dependent chemistry, the location, identity, and/or contents of cells within the chip can be monitored using brightfield or fluorescence microscopy. Cells can be stained with any stain (i.e., nucleic acid-specific stains such as SYT010; immunodetection such as Cy5-conjugated anti-CD19; etc.) that is compatible with the downstream application. This can be used, for example, to identify rare cells, i.e., cancer stem cells, within a heterogeneous cell population.

化学反应：在将细胞上样到MA006后，将检验上样到检验室(图9中的"检验通道")，释放界面阀以混合样品和检验室的内容物。对芯片进行根据所选化学反应的热循环和如果化学反应需要和/或支持，实时或在终点成像。这一程序不限于基因特异性扩增，即可使用非特异性简并引物，或可进行RNA特异性扩增。在基因特异性扩增的情形中，可利用"多路"策略同时靶向多于一个基因。化学反应是灵活的，条件是输出是测序的底物(substrate)，并且化学反应不应限于聚合酶链式反应或甚至扩增。Chemical reaction: After the cells are loaded onto the MA006, the test is loaded into the test chamber ("Test Channel" in Figure 9), and the interface valve is released to mix the sample and the contents of the test chamber. The chip is thermally cycled according to the selected chemical reaction and imaged in real time or at the endpoint if the chemical reaction requires and/or supports it. This procedure is not limited to gene-specific amplification, that is, non-specific degenerate primers can be used, or RNA-specific amplification can be performed. In the case of gene-specific amplification, a "multiplexing" strategy can be used to target more than one gene simultaneously. The chemical reaction is flexible, provided that the output is a substrate for sequencing, and the chemical reaction should not be limited to polymerase chain reaction or even amplification.

细胞操作Cell manipulation

细胞计数：亮视野成像Cell counting: bright field imaging

如下操作RAMOS细胞：RAMOS cells were manipulated as follows:

(1)收获细胞。(1) Harvest cells.

(2)在冰冷的Tris盐水BSA缓冲液中洗涤2-3X。(2) Wash 2-3X in ice-cold Tris saline BSA buffer.

(3)计数和进行适当的稀释。不同细胞密度的理论分布(泊松分布)显示在图10。(3) Count and perform appropriate dilutions. The theoretical distribution (Poisson distribution) of different cell densities is shown in Figure 10.

(4)将细胞推入MA006芯片。(4) Push the cells into the MA006 chip.

(5)通过亮视野成像。(5) Through bright field imaging.

图11A-B显示使用亮视野成像，芯片中细胞计数的结果(A)，与理论分布(B)比较。基于亮视野成像，芯片中的细胞密度接近但低于泊松分布，这一趋势在更高细胞密度时加剧。这可部分地由于芯片特征产生的"遮蔽"，其可减少利用亮视野成像可检测其中的细胞的可测量面积。Figures 11A-B show the results of cell counting in the chip using brightfield imaging (A), compared to the theoretical distribution (B). Based on brightfield imaging, the cell density in the chip is close to, but lower than, a Poisson distribution, a trend that intensifies at higher cell densities. This is partly due to "shadowing" caused by chip features, which reduces the measurable area in which cells can be detected using brightfield imaging.

细胞计数：PCR后荧光Cell counting: post-PCR fluorescence

将细胞以0.15E6/ml上样到MA006芯片并利用Cells-Direct^TM RT PCR组分、Rox和EVA green进行RT-PCR。图12A-B显示荧光细胞"鬼影"图像(A)允许比PCR前亮视野成像检测更多细胞，从而细胞密度更接近地近似泊松分布(B)。基于这些结果，如果对MA006芯片的每入口施加4000细胞(例如，4μl，1000细胞/μl)并遍布，2304(48x48)或800室的大约1/3具有单细胞。Cells were loaded onto a MA006 chip at 0.15E6/ml and RT-PCR was performed using Cells-Direct^™ RT PCR components, Rox, and EVA green. Figure 12A-B shows that fluorescent cell "ghost" images (A) allow for more cell detection than bright field imaging before PCR, resulting in a cell density that more closely approximates a Poisson distribution (B). Based on these results, if 4000 cells (e.g., 4 μl, 1000 cells/μl) are applied to each inlet of the MA006 chip and spread throughout, approximately 1/3 of 2304 (48x48) or 800 chambers have single cells.

更特异性的方法More specific methods

可使用的用于检测芯片中细胞的更特异性的方法包括，例如，使用细胞膜透性核酸染色剂和/或用抗体检测细胞特异性表面标志物。如此，例如，可如下操作RAMOS细胞：More specific methods for detecting cells in the chip that can be used include, for example, the use of cell membrane permeable nucleic acid stains and/or the use of antibodies to detect cell-specific surface markers. Thus, for example, RAMOS cells can be manipulated as follows:

(1)收获细胞。(1) Harvest cells.

(2)在冰冷的Tris盐水BSA缓冲液中洗涤2-3X。(2) Wash 2-3X in ice-cold Tris saline BSA buffer.

(3)用Syto10 DNA染色剂和/或Cy5-标记的抗CD19抗体染色。(3) Staining with Syto10 DNA stain and/or Cy5-labeled anti-CD19 antibody.

(4)在冰冷的Tris盐水BSA缓冲液中洗涤2-3X。(4) Wash 2-3X in ice-cold Tris saline BSA buffer.

(5)计数并进行适当的稀释。(5) Count and make appropriate dilutions.

(6)将细胞推入MA006芯片。(6) Push the cells into the MA006 chip.

(7)成像。(7) Imaging.

在图13中，这些更特异性的方法的结果对1E6/ml的细胞密度显示。图14A显示RT-PCR前核酸染色剂(Syto10 DNA染色剂)与RT-PCR后鬼影图像(细胞鬼影)的比较，图14B显示Syto10不抑制GAPDH的RT-PCR。芯片中细胞检测的工作流可包括用DNA染色剂和/或抗体染色细胞，随后RT-PCR前计数和然后RT-PCR后计数细胞鬼影作为备份(back-up)。In Figure 13, the results of these more specific methods are shown for a cell density of 1E6/ml. Figure 14A shows a comparison of a nucleic acid stain (Syto10 DNA stain) before RT-PCR with a ghost image (cell ghost) after RT-PCR, and Figure 14B shows that Syto10 does not inhibit RT-PCR of GAPDH. The workflow for cell detection on the chip can include staining cells with DNA stains and/or antibodies, followed by counting cells before RT-PCR and then counting cell ghosts after RT-PCR as a backup.

化学反应：一步基因特异性RT-PCRChemistry: One-step gene-specific RT-PCR

研究了不同的化学反应以寻找在MA006芯片中转化细胞中的基因特异性RNA为扩增子的有效化学反应。将细胞推入细胞通道的Tris盐水BSA(0.5μg/ml)缓冲液中。上样到检验通道的试剂包括：Different chemical reactions were investigated to identify an effective reaction for converting gene-specific RNA from cells into amplicons on the MA006 chip. Cells were introduced into the cell channel in a Tris-saline BSA (0.5 μg/ml) buffer. Reagents loaded into the assay channel included:

引物(500nM终浓度)Primers (500 nM final concentration)

CellsDirect^TM一步qRT-PCR试剂盒组分(从Life Technologies,Foster City,CA可得)CellsDirect^™ One-Step qRT-PCR Kit Components (available from Life Technologies, Foster City, CA)

反应混合物Reaction mixture

酶混合物：Superscripte III+Platinum Taq聚合酶Enzyme mixture: Superscripte III + Platinum Taq polymerase

缓冲液buffer

RoxRox

EVA GreenEVA Green

上样试剂-AA或GE(从Fluidigm Corp.,South San Francisco,CA可得)以阻止被PDMS非特异性吸收("耗竭效应")并溶解细胞。Loading reagent—AA or GE (available from Fluidigm Corp., South San Francisco, CA)—was used to prevent nonspecific adsorption to PDMS ("depletion effect") and to lyse cells.

带有或不带有AA或GE上样试剂地进行GAPDH的RT-PCR。结果显示，两种上样试剂都抑制RT-PCR。上样试剂包含：Prionex(AA)或BSA(GE)和0.5％Tween-20。GAPDH的RT-PCR在Prionex或BSA存在下进行。发现Prionex但不是BSA抑制RT-PCR。GAPDH的RT-PCR在0.5％Tween 20或0.5％NP40(后者是细胞溶解剂)存在下进行。这一研究的结果显示在图15。0.5％Tween 20和0.5％NP40都不显著抑制GAPDH的RT-PCR。RT-PCR for GAPDH was performed with or without AA or GE loading reagents. The results showed that both loading reagents inhibited RT-PCR. The loading reagents contained: Prionex (AA) or BSA (GE) and 0.5% Tween-20. RT-PCR for GAPDH was performed in the presence of Prionex or BSA. Prionex, but not BSA, was found to inhibit RT-PCR. RT-PCR for GAPDH was performed in the presence of 0.5% Tween 20 or 0.5% NP40 (the latter is a cell lysis agent). The results of this study are shown in Figure 15. Neither 0.5% Tween 20 nor 0.5% NP40 significantly inhibited RT-PCR for GAPDH.

为了确定为来自细胞的GAPDH的RT-PCR开发的反应条件将允许以不同的水平表达的其他基因的RT-PCR，覆盖一定范围表达水平的11个基因的RT-PCR用10ng/μl RNA和上述试剂进行，除了0.5％NP40代替AA/GE上样试剂。热循环方案是：50℃30分钟；55℃30分钟；95℃2分钟；然后45个循环：95℃15秒、60℃30秒、和72℃60秒。图16中显示在MA006芯片中进行的这些11个基因的标准曲线扩增。这些结果证明，CellsDirect^TM一步qRT-PCR试剂盒可与0.5％NP40(用于细胞溶解和阻止芯片中的耗竭效应)一起使用来在MA006芯片中将细胞中基因特异性的RNA转化为扩增子。To confirm that the reaction conditions developed for RT-PCR of GAPDH from cells would allow for RT-PCR of other genes expressed at varying levels, RT-PCR of 11 genes covering a range of expression levels was performed using 10 ng/μl RNA and the above reagents, except that 0.5% NP40 was used instead of the AA/GE loading reagent. The thermal cycling protocol was: 50°C for 30 minutes; 55°C for 30 minutes; 95°C for 2 minutes; followed by 45 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 60 seconds. Figure 16 shows the standard curve amplification of these 11 genes performed in the MA006 chip. These results demonstrate that the CellsDirect^™ One-Step qRT-PCR Kit can be used with 0.5% NP40 (for cell lysis and to prevent depletion effects in the chip) to convert gene-specific RNA from cells into amplicons in the MA006 chip.

测序Sequencing

为了帮助测序在MA006芯片中产生的基因特异性扩增子，采用条形码化方法以区分来自不同的室(例如，细胞)的扩增子。更具体地，采用四引物、组合条形码化方法以将两个条形码的组合放到每个扩增子的任一末端上。这一方法在图17中图解显示。内侧引物包含靶特异性部分(正向引物中的"TS-F"和反向引物中的"TS-R")、条形码核苷酸序列("bc2")、和不同的核苷酸标签。外侧引物包含标签特异性部分("CS1"和"CS2")、不同的条形码核苷酸序列("bc1")、和用于测序引物的引物结合位点("A"和"B")。图18A-B图解4引物条形码化如何可在芯片诸如MA006上进行。在芯片上用内侧引物进行扩增，其中每行的室具有拥有相同的条形码的相同的内侧引物对。来自每列的室的反应产物可作为池收获，并对每个池利用不同的外侧引物对进行扩增。这一扩增产生在扩增子的任一末端具有独特地辨识在其中进行初始扩增的室(以行和列)的条形码组合的扩增子。测序反应产物并确定每个反应区室的每个序列的读取的数目。这一确定对RAMOS细胞和对脾RNA进行。图19显示对获得的结果的比较，表示为与总RNA的读取的数目相比，对每个基因特异性扩增子的读取的数目(红)。如从该图明显的，与在总RNA中观察到的相比，这些RNA的代表当在单独细胞中测量时是不同的。In order to help sequence the gene-specific amplicons produced in the MA006 chip, a barcoding method is used to distinguish amplicons from different chambers (e.g., cells). More specifically, a four-primer, combined barcoding method is used to place a combination of two barcodes on either end of each amplicon. This method is illustrated in Figure 17. The inner primers include a target-specific portion ("TS-F" in the forward primer and "TS-R" in the reverse primer), a barcode nucleotide sequence ("bc2"), and different nucleotide tags. The outer primers include a tag-specific portion ("CS1" and "CS2"), different barcode nucleotide sequences ("bc1"), and primer binding sites ("A" and "B") for sequencing primers. Figure 18A-B illustrates how 4-primer barcoding can be performed on a chip such as MA006. Amplification is performed on the chip with an inner primer, wherein the chambers of each row have the same inner primer pair with the same barcode. The reaction product from the chamber of every column can be used as pond crops, and different outside primers are utilized to amplify in each pond. This amplification produces an amplicon that has the barcode combination of the chamber (with row and column) that uniquely identifies the initial amplification therein at either end of the amplicon. Sequencing reaction product also determines the number of readings of each sequence of each reaction compartment. This determination is carried out to RAMOS cells and to spleen RNA. Figure 19 shows the comparison of the result obtained, is expressed as compared with the number of readings of total RNA, to the number (red) of readings of each gene-specific amplicon. As obvious from the figure, compared with observed in total RNA, the representatives of these RNAs are different when measured in individual cells.

实施例6Example 6

基于尺寸的微流体单颗粒捕获Size-based single-particle capture in microfluidics

当悬液流经微流体装置时从悬液离散地捕获(discretely capturing)单细胞的一个方法是界定以以下方式导引颗粒(诸如细胞或珠)的悬液流过捕获位点的微流体几何形状：捕获位点捕捉单颗粒、有效捕获单颗粒(例如，捕获经过捕获位点附近的颗粒的概率高)、和/或导引围绕捕获位点的剩余悬液。几何形状可以是基于尺寸的，即，捕获位点仅仅足够大以包含一个颗粒(且不多于)，但仍允许无颗粒悬液以适当低的流体阻抗流经该位点，从而空的捕获位点将导引颗粒的流朝向它而不是围绕它。这一目的可通过使用引流沟来实现。另外的几何形状还可以以下方式集中颗粒的流：为了成功捕获的高概率，增加颗粒去到足够接近捕获位点的可能性。对这些几何形状的变化集中在控制围绕捕获位点和引流沟、包括引流沟自身的流体的流阻，以及改变集中几何形状的孔以试图定位接近捕获位点的颗粒的流。图20A-B示例带有捕获特征和引流沟的捕获位点。图A显示无阻碍物以集中流的位点，而图B显示带有阻碍物的位点。另外的捕获位点设计显示在图21。One approach to discretely capturing a single cell from a suspension as it flows through a microfluidic device is to define a microfluidic geometry that directs the flow of a suspension of particles (such as cells or beads) through a capture site in such a way that the capture site captures a single particle, effectively captures a single particle (e.g., the probability of capturing particles passing near the capture site is high), and/or directs the remaining suspension around the capture site. The geometry can be size-based, i.e., the capture site is only large enough to contain one particle (and no more), but still allows a particle-free suspension to flow through the site with a suitably low fluid impedance so that an empty capture site will direct the flow of particles toward it rather than around it. This goal can be achieved by using drainage grooves. Additional geometries can also focus the flow of particles in such a way that the probability of particles getting close enough to the capture site is increased for a high probability of successful capture. Variations on these geometries focus on controlling the flow resistance of the fluid around the capture site and the drainage grooves, including the drainage grooves themselves, and on changing the apertures of the focusing geometry to attempt to locate the flow of particles close to the capture site. Figures 20A-B illustrate capture sites with capture features and drainage grooves. Panel A shows a site without obstructions to focus flow, while panel B shows a site with obstructions. Additional capture site designs are shown in Figure 21.

实施例7Example 7

基于表面标志物捕获颗粒Capture particles based on surface markers

微流体结构中的单细胞研究要求分离单独细胞到单独反应区隔(室、液滴、颗粒)中。有限稀释是实现这一分离的一个方法。细胞被以平均每区隔小于一个细胞的浓度上样，并以泊松统计学描述的方式分配到那些区隔中。另一种方法是依赖于机械陷阱来捕获细胞。这些陷阱设计为捕获给定尺寸范围的细胞(参见实施例6)。这导致从群体偏置地选择在该尺寸范围中的细胞。Single cell studies in microfluidic structures require the separation of individual cells into separate reaction compartments (chambers, droplets, particles). Limiting dilution is one method for achieving this separation. Cells are loaded at an average concentration of less than one cell per compartment and distributed into those compartments in a manner described by Poisson statistics. Another method relies on mechanical traps to capture cells. These traps are designed to capture cells of a given size range (see Example 6). This results in a biased selection of cells in that size range from the population.

对于一些应用，理想的捕获方法将利用在细胞表面上表达的生物标志物。抗体可以在微流体阵列上的特定位置排列，尽管取决于微流体阵列的结构，这一方法可能不简单。For some applications, the ideal capture method will utilize biomarkers expressed on the cell surface. Antibodies can be arrayed at specific locations on a microfluidic array, although this approach may not be straightforward depending on the structure of the microfluidic array.

这一实施例描述了基于最初捕获在微流体装置中特定位置的单个、亲和性试剂包被的珠来捕获单颗粒(例如，细胞)的方法。在捕获位点开口处被该珠占据的表面积提供了细胞结合可及的亲和性试剂的确定表面。珠尺寸和捕获位点可被选择/设计使得单细胞结合到珠后，珠的其余可及表面积被先结合的细胞空间封闭。尺寸适当的珠捕获位点的选择还提供宽范围细胞尺寸的捕获。只要细胞大于暴露的捕获面积，并表达适当的表面标志物或亲和性试剂的结合伴侣，捕获该细胞就应是可能的。This embodiment describes a method for capturing single particles (e.g., cells) based on single, affinity reagent-coated beads initially captured at specific locations in a microfluidic device. The surface area occupied by the beads at the capture site opening provides a definite surface for cell binding to accessible affinity reagents. Bead size and capture site can be selected/designed so that after a single cell is bound to the beads, the remaining accessible surface area of the beads is spatially closed by the cells that were previously bound. The selection of appropriately sized bead capture sites also provides for the capture of a wide range of cell sizes. As long as the cell is larger than the exposed capture area and expresses a suitable surface marker or binding partner for the affinity reagent, it should be possible to capture the cell.

捕获结构可被设计为使得细胞接触表面标志物的概率最大化。例如，一个或多个通道壁上的阻碍物可用于引导珠朝向捕获特征。参见图22A的示例性的捕获特征/阻碍物组合。捕获特征的表现可通过调整一个或多个变量来调整，所述变量包括阻碍物的角度、阻碍物与捕获位点的距离、阻碍物的长度、捕获特征的尺寸和形状、捕获特征中引流沟的尺寸(如果存在)。参见图22B和C，示例捕获特征/阻碍物组合的变量和表现。在图22B中，通道壁上的阻碍物用于引导珠朝向捕获特征。在图22C中，捕获特征与通道壁上的阻碍物配对；单独的捕获特征/阻碍物组合可位于交替的壁上以将流集中朝向邻近的捕获特征/阻碍物组合。这些组合可位于在使用中可分隔(例如，利用阀)的位点以形成分别的反应区室。The capture structure can be designed to maximize the probability of cell contact surface markers. For example, one or more obstructions on the channel wall can be used to guide beads toward the capture feature. See the exemplary capture feature/obstruction combination of Figure 22A. The performance of the capture feature can be adjusted by adjusting one or more variables, including the angle of the obstruction, the distance between the obstruction and the capture site, the length of the obstruction, the size and shape of the capture feature, and the size of the drainage groove in the capture feature (if present). See Figures 22B and C, for example, the variables and performance of the capture feature/obstruction combination. In Figure 22B, the obstruction on the channel wall is used to guide beads toward the capture feature. In Figure 22C, the capture feature is paired with the obstruction on the channel wall; a separate capture feature/obstruction combination can be located on alternating walls to concentrate the flow toward the adjacent capture feature/obstruction combination. These combinations can be located at sites that can be separated (e.g., using valves) in use to form separate reaction chambers.

图23A和B示例(以简化形式，缺少阻碍物)利用捕获特征捕捉单个、亲和性试剂包被的珠的策略，该珠随后展示亲和性试剂(例如，抗体)以捕获单颗粒(例如，细胞)。在图23A-1中，流在包含捕获特征的通道中开始。在图A-2中，抗体结合的珠流向捕获特征，直到珠安顿在捕获特征中，如图A-3所示。然后洗涤通道以去除未捕获的珠。随后，如图23B-1所示，带有抗体结合的细胞表面标志物的细胞流入包含捕获的珠的通道。图B-2示例带有标志物的细胞如何与被捕获的珠展示的抗体相互作用和结合。展示区域的尺寸为使得结合的细胞将经由空间阻塞抑制其他细胞与捕获的珠相互作用，从而仅一个细胞结合每个捕获的珠。如图B-3所示，然后洗涤通道以去除未结合的细胞，在每个捕获位点留下一个固定的细胞。Figure 23A and B examples (in simplified form, lacking obstructions) utilize capture features to capture a single, affinity reagent coated bead strategy, which then displays an affinity reagent (e.g., antibody) to capture a single particle (e.g., cell). In Figure 23A-1, flow begins in a channel comprising a capture feature. In Figure A-2, the antibody-bound beads flow to the capture feature until the beads settle in the capture feature, as shown in Figure A-3. The wash channel is then used to remove uncaptured beads. Subsequently, as shown in Figure 23B-1, cells with antibody-bound cell surface markers flow into the channel comprising the captured beads. Figure B-2 illustrates how cells with markers interact and combine with the antibodies displayed by the captured beads. The size of the display area is such that the combined cells will inhibit other cells from interacting with the captured beads via spatial obstruction, thereby only one cell combines each captured bead. As shown in Figure B-3, the wash channel is then used to remove unbound cells, leaving a fixed cell at each capture site.

实施例8Example 8

用于细胞捕获("CCap")的微流体装置Microfluidic Devices for Cell Capture ("CCap")

图24A显示设计为在离散的位置(龛)捕获单细胞的微流体装置的简图。流设计为在龛上方比经过溢流通道的强。龛包含小的缺口(～3μm高)。参见图24B。当细胞进入龛时，它封闭龛并阻止任何更多的流进入龛。流穿过到下一个未被占据的龛，直到它也被细胞封闭。理论上，在细胞穿过溢流通道并离开废弃之前，每个龛应捕获一个细胞。参考图24C-F的更多细节，缓冲液入口与细胞入口汇集，从而迫使细胞向最接近一系列横向细胞捕获通道的给料通道的一侧。参见图24D。横向细胞捕获通道的阻力低于细胞溢流通道的，以引导细胞流优先进入龛而不是进入细胞溢流通道。参见图24E。如图24F所示，每个龛足够大以捕获仅一个细胞。龛缺口足够小，使得细胞在操作压力/流水平被捕获。如果后者过高和/或龛缺口过大，细胞可变形并被推动穿过龛缺口。龛中细胞的存在升高了该特定回路的阻力，因此流被导向无细胞的回路。图24G显示实际装置，捕获的人类脐静脉内皮细胞(HUVEC)位于龛中。Figure 24A shows a simplified diagram of a microfluidic device designed to capture single cells at discrete locations (niches). Flow is designed to be stronger over the niches than through the overflow channels. The niches contain small gaps (~3 μm high). See Figure 24B. When a cell enters a niche, it seals the niche and prevents further flow from entering. Flow passes to the next unoccupied niche until it, too, is sealed by a cell. Theoretically, each niche should capture one cell before the cell passes through the overflow channels and exits the waste stream. Referring to Figures 24C-F for more detail, the buffer inlet converges with the cell inlet, forcing the cell toward the side of the feed channel closest to a series of lateral cell-capturing channels. See Figure 24D. The lateral cell-capturing channels have lower resistance than the cell overflow channels, directing the cell flow preferentially into the niches rather than into the cell overflow channels. See Figure 24E. As shown in Figure 24F, each niche is large enough to capture only one cell. The niche gaps are small enough to allow cells to be captured at the operating pressure/flow level. If the latter is too high and/or the niche gaps are too large, cells can deform and be pushed through the niche gaps. The presence of cells in the niche increases the resistance of that particular circuit, so flow is directed towards the cell-free circuit. Figure 24G shows the actual device, with captured human umbilical vein endothelial cells (HUVECs) located in the niche.

实施例9Example 9

利用48.48 ACCESS ARRAY^TM IFC、用于Illumina测序仪的双向DNA测序扩增子加标Bidirectional DNA Sequencing Amplicon Spiking for Illumina Sequencers Using the 48.48 ACCESS ARRAY^™ IFC签-方案1Sign-Option 1

引言introduction

以下方案概述了对于在ACCESS ARRAY^TM System上已经产生的扩增子文库，在Illumina Genome GAII、HiSeq和MiSeq测序仪上的双向测序策略。这一方案的目的是以单个读取测序运行测序PCR产物的两个末端。在标准的4引物扩增子加标签方法(参见实施例6)中，将加标签的靶特异性(TS)引物对与包含条形码序列(BC)的样品特异性引物对和Illumina测序仪所用的衔接子序列(PE1和PE2，图25A)组合。此处，在双向测序扩增子加标签策略中，不同地，将加标签的靶特异性引物对与两组样品特异性引物对组合。样品特异性引物对包含共有序列标签CS1或CS2，以两种排列附带有Illumina衔接子序列(PE1和PE2，图25B)。这一方法仅要求一组靶特异性引物对，而样品特异性条形码引物是通用的，可用在多个实验中。The following scheme outlines the bidirectional sequencing strategy on Illumina Genome GAII, HiSeq and MiSeq sequencers for the amplicon libraries that have been generated on the ACCESS ARRAY^™ System. The purpose of this scheme is to run sequencing of the two ends of the PCR product with a single read. In the standard 4-primer amplicon tagging method (see Example 6), the target-specific (TS) primers of the tagging are combined with the sample-specific primers comprising the barcode sequence (BC) and the adapter sequences (PE1 and PE2, Figure 25A) used by the Illumina sequencer. Here, in the bidirectional sequencing amplicon tagging strategy, differently, the target-specific primers of the tagging are combined with two groups of sample-specific primers. The sample-specific primers are to comprise a consensus sequence tag CS1 or CS2, with two arrangements accompanied by an Illumina adapter sequence (PE1 and PE2, Figure 25B). This method only requires one group of target-specific primers to be right, and the sample-specific barcode primers are universal and can be used in multiple experiments.

双向测序扩增子加标签产生每个靶区域两个类型的PCR产物：一个PCR产物允许测序靶区域的5'末端(产物A)和一个PCR产物允许测序靶区域的3'末端(产物B)。因为两个PCR产物同时存在于流动小室，一个测序读取产生靶区域两个末端的序列信息。这一策略与末端配对的测序(实施例6)之间的主要差异在于，5'读取和3'读取不是来源于相同的簇，即，来自相同的模板分子。相反，衍生了模板群体的平均值。Bidirectional sequencing amplicon tagging generates two types of PCR products per target region: one PCR product that allows sequencing of the 5' end of the target region (product A) and one PCR product that allows sequencing of the 3' end of the target region (product B). Because both PCR products are present simultaneously in the flow cell, a single sequencing read generates sequence information for both ends of the target region. The main difference between this strategy and paired-end sequencing (Example 6) is that the 5' read and the 3' read are not derived from the same cluster, i.e., from the same template molecule. Instead, an average of the template population is derived.

多个靶序列的扩增可在加入双向条形码之前进行。简言之，该方案采用两步方法：ACCESS ARRAY IFC上的PCR在仅多路、加标签的、靶特异性引物存在下运行。然后收获的PCR产物池用作用样品特异性条形码引物的第二PCR的模板。在独立PCR反应中加入两组条形码引物，如下所述。Amplification of multiple target sequences can be performed before adding bidirectional barcodes. Briefly, the protocol uses a two-step approach: PCR on the ACCESS ARRAY IFC is run in the presence of only multiplexed, tagged, target-specific primers. The pool of harvested PCR products is then used as the template for a second PCR using sample-specific barcoded primers. Two sets of barcoded primers are added in separate PCR reactions, as described below.

将样品特异性条形码引物对分离到两个分别的PCR反应中(图26；还参见表1)。Sample-specific barcoded primer pairs were separated into two separate PCR reactions (Figure 26; see also Table 1).

表1.分离的-引物PCR策略中使用的条形码引物.Table 1. Barcoded primers used in the split-primer PCR strategy.

引物Primers序列sequencePE1-CS1PE1-CS15’-AATGATACGGCGACCACCGAGATCTACACTGACGACATGGTTCTACA-3'(SEQ ID N0：9)5’-AATGATACGGCGACCACCGAGATCTACACTGACGACATGGTTCTACA-3’ (SEQ ID N0: 9)PE2-BC-CS2PE2-BC-CS25’-CAAGCAGAAGACGGCATACGAGAT-[BC]-TACGGTAGCAGAGACTTGGTCT-3'(SEQ ID NO：10)5’-CAAGCAGAAGACGGCATACGAGAT-[BC]-TACGGTAGCAGAGACTTGGTCT-3’ (SEQ ID NO: 10)PE1-CS2PE1-CS25’-AATGATACGGCGACCACCGAGATCTTACGGTAGCAGAGACTTGGTCT-3’(SEQ ID NO：11)5’-AATGATACGGCGACCACCGAGATCTTACGGTAGCAGAGACTTGGTCT-3’ (SEQ ID NO: 11)PE2-BC-CS1PE2-BC-CS15’-CAAGCAGAAGACGGCATACGAGAT-[BC]-ACACTGACGACATGGTTCTACA-3'(SEQ ID N0：12)5’-CAAGCAGAAGACGGCATACGAGAT-[BC]-ACACTGACGACATGGTTCTACA-3’ (SEQ ID N0: 12)

条形码化PCR后，合并5'反应和3'反应二者的PCR产物并将该产物用作在流动小室上形成簇的模板。由于两个PCR产物类型都存在并在流动小室上形成簇，CS1和CS2测序引物的等摩尔混合物允许同时测序两个PCR产物类型(图27)。类似地，具有CS1rc和CS2rc测序引物的等摩尔混合物的索引读取允许同时测序两个PCR产物类型的条形码。After barcoding PCR, the PCR products of both the 5' and 3' reactions were combined and used as templates for cluster formation on the flow cell. Since both PCR product types were present and formed clusters on the flow cell, an equimolar mixture of CS1 and CS2 sequencing primers allowed simultaneous sequencing of both PCR product types (Figure 27). Similarly, an index read with an equimolar mixture of CS1rc and CS2rc sequencing primers allowed simultaneous sequencing of the barcodes of both PCR product types.

可查阅IFC Controller for ACCESS ARRAY^TM System User Guide(PN 68000157)作为用于这一方案的参考。可查阅Illumina网址的最新方案、试剂和目录号信息。The IFC Controller for ACCESS ARRAY^™ System User Guide (PN 68000157) is available as a reference for this protocol. The latest protocol, reagent, and catalog number information is available on the Illumina website.

制备和测序扩增子Preparation and sequencing of amplicons

以下试剂用于这一方案并储存在-20℃：FastStart高保真PCR System,dNTPack(Roche,PN 04-738-292-001)；20X ACCESS ARRAY^TM上样试剂(Fluidigm,PN 100-0883)；带有通用标签(CS1正向标签、CS2反向标签)的靶特异性引物对，包括50μM CS1-加标签的TS正向引物和50μM CS2-加标签的TS反向引物；和用于Illumina GAII、HiSeq和MiSeq测序仪的双向384条形码试剂盒(Fluidigm,PN 100-3771)。另外的试剂储存在4℃，包括：AgilentDNA 1000试剂盒试剂(Agilent,PN 5067-1504)；和1 X ACCESS ARRAY^TM收获溶液(Fluidigm,PN 100-1031)。其他试剂储存在室温，包括PCR Certified Water(Teknova,PNW330)；DNA悬浮缓冲液(10 mM Tris HCI,0.1 mM EDTA,pH8.0)(Teknova,PN T0221)；和Agilent DNA 1000芯片(包括在Agilent DNA 1000 DNA试剂盒中)(Agilent)。The following reagents were used for this protocol and stored at −20°C: FastStart High-Fidelity PCR System, dNTPack (Roche, PN 04-738-292-001); 20X ACCESS ARRAY^™ loading reagent (Fluidigm, PN 100-0883); target-specific primer pairs with universal tags (CS1 forward tag, CS2 reverse tag), including 50 μM CS1-tagged TS forward primer and 50 μM CS2-tagged TS reverse primer; and a bidirectional 384 barcoding kit for Illumina GAII, HiSeq, and MiSeq sequencers (Fluidigm, PN 100-3771). Additional reagents were stored at 4°C, including: Agilent DNA 1000 kit reagents (Agilent, PN 5067-1504); and 1 X ACCESS ARRAY^™ harvest solution (Fluidigm, PN 100-1031). Other reagents were stored at room temperature, including PCR Certified Water (Teknova, PN W330); DNA suspension buffer (10 mM Tris HCl, 0.1 mM EDTA, pH 8.0) (Teknova, PN T0221); and Agilent DNA 1000 chip (included in the Agilent DNA 1000 DNA kit) (Agilent).

以下设备和消耗品用于这一方案：1.5mL或2mL微量离心管；带有用于2 mL管的转子的微量离心机；带有用于0.2mL PCR管条(tube strip)的转子的微量离心机；带有板架(plate carrier)的离心机；Agilent 2100BioAnalyzer(Agilent)；96孔反应板；MicroAmpClear Adhesive Film(Applied Biosystems,PN 4306311)；IFC Controller AX(2倍量，PCR前和PCR后)(Fluidigm)；FC1 Cycler(Fluidigm)；48.48 ACCESS ARRAY^TM IFC s(Fluidigm)；和Control Line Fluid Syringes(Fluidigm,PN 89000020)。The following equipment and consumables were used for this protocol: 1.5 mL or 2 mL microcentrifuge tubes; a microcentrifuge with a rotor for 2 mL tubes; a microcentrifuge with a rotor for 0.2 mL PCR tube strips; a centrifuge with a plate carrier; an Agilent 2100 BioAnalyzer (Agilent); 96-well reaction plates; MicroAmpClear Adhesive Film (Applied Biosystems, PN 4306311); an IFC Controller AX (2x volume, pre-PCR and post-PCR) (Fluidigm); an FC1 Cycler (Fluidigm); 48.48 ACCESS ARRAY^™ IFCs (Fluidigm); and Control Line Fluid Syringes (Fluidigm, PN 89000020).

在ACCESS ARRAY^TM IFC上的多路PCR按照在Fluidigm ACCESS ARRAY^TM Systemfor Illumina Platform User Guide中的Chapter 6-Multiplex PCR on the 48.48ACCESS ARRAY^TM IFC中详述的说明进行。Multiplex PCR on the ACCESS ARRAY^™ IFC was performed according to the instructions detailed in Chapter 6 - Multiplex PCR on the 48.48 ACCESS ARRAY^™ IFC in the Fluidigm ACCESS ARRAY^™ System for Illumina Platform User Guide.

条形码化PCR按照在Fluidigm ACCESS ARRAY System for Illumina PlatformUser Guide中的Chapter 6-Attaching Sequence Tags and Sample Barcodes中详述的说明进行。收获的PCR产物池的100X稀释物用作二个而不是一个条形码化PCR反应的模板：一个反应产生的PCR产物A允许测序靶区域的5'末端，另一个反应产生的PCR产物B允许测序靶区域的3'末端。反应的设置与Fluidigm ACCESS ARRAY System for Illumina PlatformUser Guide中的"Attaching Sequence Tags and Sample Barcodes"相同。然而，样品预混合物主混合物(Sample Pre-Mix Master Mix)的量加倍以补偿孔数目的增加。在第二PCR完成后，合并PCR产物A和PCR产物B池，随后测序。Fluidigm ACCESS ARRAY^TM System forIllumina Platform User Guide的第8章提供描述PCR后产物文库纯化和定量的方法。Barcoding PCR was performed according to the instructions detailed in Chapter 6 - Attaching Sequence Tags and Sample Barcodes in the Fluidigm ACCESS ARRAY System for Illumina Platform User Guide. A 100X dilution of the harvested PCR product pool was used as a template for two barcoding PCR reactions instead of one: PCR product A produced in one reaction allowed sequencing of the 5' end of the target region, and PCR product B produced in the other reaction allowed sequencing of the 3' end of the target region. The reaction setup was the same as "Attaching Sequence Tags and Sample Barcodes" in the Fluidigm ACCESS ARRAY System for Illumina Platform User Guide. However, the amount of Sample Pre-Mix Master Mix was doubled to compensate for the increase in the number of wells. After the second PCR was complete, the PCR product A and PCR product B pools were combined and subsequently sequenced. Chapter 8 of the Fluidigm ACCESS ARRAY^™ System for Illumina Platform User Guide provides a method describing post-PCR product library purification and quantification.

这一实施例的其余部分提供该方案中使用的测序工作流。The remainder of this example provides the sequencing workflow used in this protocol.

以下制备试剂的说明预期是与Illumina TruSeq测序试剂一起使用。Fluidigm试剂FL1和FL2分别包含CS1和CS2测序和索引引物的等摩尔混合物。FL1是测序引物，包含各50μM的CS1和CS2引物。FL2是索引引物，包含各50μM的CS1rc和CS2rc引物。这些引物的序列显示在表2。The following instructions for preparing reagents are intended for use with Illumina TruSeq sequencing reagents. Fluidigm reagents FL1 and FL2 contain equimolar mixtures of CS1 and CS2 sequencing and indexing primers, respectively. FL1 is a sequencing primer containing 50 μM each of CS1 and CS2 primers. FL2 is an indexing primer containing 50 μM each of CS1rc and CS2rc primers. The sequences of these primers are shown in Table 2.

表2引物和序列Table 2 Primers and sequences

引物Primers序列sequenceCS1CS15’-ACACTGACGACATGGTTCTACA-3'(SEQ ID NO：13)5’-ACACTGACGACATGGTTCTACA-3’ (SEQ ID NO: 13)CS2CS25’-TACGGTAGCAGAGACTTGGTCT-3’(SEQ ID NO:14)5’-TACGGTAGCAGAGACTTGGTCT-3’(SEQ ID NO:14)CS1rcCS1rc5’-TGTAGAACCATGTCGTCAGTGT-3’(SEQ ID NO：15)5’-TGTAGAACCATGTCGTCAGTGT-3’ (SEQ ID NO: 15)CS2rcCS2rc5’-AGACCAAGTCTCTGCTACCGTA-3’(SEQ ID NO：16)5’-AGACCAAGTCTCTGCTACCGTA-3’ (SEQ ID NO: 16)

测序引物HP6/FL1通过在无DNA酶、RNA酶的0.5mL微量离心管中在TruSeq试剂HP6中稀释Fluidigm试剂FL1(其包含定制的测序引物)到终浓度0.25μM来制备，如表3所示。混合后涡旋引物以确保完全混合。Sequencing primer HP6/FL1 was prepared by diluting Fluidigm reagent FL1 (which contains custom sequencing primers) in TruSeq reagent HP6 to a final concentration of 0.25 μM in a DNase- and RNase-free 0.5 mL microcentrifuge tube, as shown in Table 3. The primers were vortexed after mixing to ensure complete mixing.

表3制备HP6/FL 1(每mL)的说明Table 3 Instructions for preparing HP6/FL 1 (per mL)

试剂Reagents体积volumeTruSeq试剂HP6TruSeq Reagent HP6995μL995μLFL1FL15μL5μL共common1000μL1000μL

索引引物HP8/FL2通过在无DNA酶、RNA酶的0.5ml微量离心管中在Truseq试剂HP8中稀释Fluidigm试剂FL2(其包含定制的索引引物)到终浓度0.25μM来制备，如表4所示。混合后涡旋引物以确保完全混合。Index primer HP8/FL2 was prepared by diluting Fluidigm reagent FL2 (which contains the custom index primer) in Truseq reagent HP8 to a final concentration of 0.25 μM in a DNase-, RNase-free 0.5 ml microcentrifuge tube as shown in Table 4. The primers were vortexed after mixing to ensure complete mixing.

表4制备HP8/FL2(每mL)的说明Table 4 Instructions for preparing HP8/FL2 (per mL)

试剂Reagents体积volumeTruSeq试剂HP8TruSeq Reagents HP8995μL995μLFL2FL25μL5μL共common1000μL1000μL

簇利用Illumina cBot^TM User Guide、Illumina Cluster Station User Guide或Illumina MiSeq User Guide中的详细说明产生。为了杂交测序引物，测序引物试剂HP6/FL1用于第一读取。Clusters were generated using the detailed instructions in the Illumina cBot^™ User Guide, Illumina Cluster Station User Guide, or Illumina MiSeq User Guide.For hybridization of sequencing primers, sequencing primer reagent HP6/FL1 was used for the first read.

根据制造商的说明制备测序试剂并上样到测序仪。对于读取1，遵循制造商提供的说明来进行多路的单读取测序运行。Prepare sequencing reagents according to the manufacturer's instructions and load onto the sequencer. For read 1, follow the manufacturer's instructions for a multiplexed single-read sequencing run.

对于索引读取，索引试剂HP7/FL2被代替，而不是HP7试剂。Fluidigm双向引物文库中使用的条形码序列设计为使得即使存在测序误差时仍可区分它们。随着平行运行更多样品，区分条形码序列所需的索引读取的长度明显增加。索引读取的建议描述在表5。For index reads, the index reagent HP7/FL2 was substituted instead of the HP7 reagent. The barcode sequences used in the Fluidigm bidirectional primer library were designed to allow for discrimination even in the presence of sequencing errors. As more samples are run in parallel, the length of the index reads required to discriminate between barcode sequences increases significantly. Recommendations for index reads are described in Table 5.

表5索引读取建议Table 5 Index read recommendations

每泳道的样品数目Number of samples per lane1-961-9697-38497-384385-1920385-1920索引读取的长度Length of index reads6个碱基6 bases8个碱基8 bases10个碱基10 bases

制备测序运行时，按照表5中的指导调整索引读取的长度。确保上样到测序仪的测序试剂的体积足够用于索引循环。根据制造商的建议实现这些改变。When preparing for a sequencing run, adjust the length of the index reads according to the guidelines in Table 5. Ensure that the volume of sequencing reagents loaded onto the sequencer is sufficient for the index cycle. Implement these changes according to the manufacturer's recommendations.

实施例10Example 10

利用微流体装置、用于双向Illumina测序、允许回收扩增产物的将靶核酸加标签Tagging of target nucleic acids using a microfluidic device for bidirectional Illumina sequencing, allowing recovery of amplification products的详细程序Detailed procedures

设计394个引物对以从基因BRCA1、BRCA2、PTEN、PI3KCA、APC、EGFR、TP53中PCR扩增外显子(参见下表6)。正向引物附加Tag8序列，反向引物附加Tag5序列。394个引物被布置为48组，每组包含平均大约8个引物对，以0.05％Tween-20中每个引物1μM的浓度。样品混合物从48个细胞系基因组DNA样品(参见下表7)通过加入1μl样品(50 ng/ul)到3μl样品预混合物制备，样品预混合物包含1U Roche Faststart HiFi聚合酶、1x缓冲液、100μM dNTP、4.5mM MgCl₂、5％DMSO和1xACCESS ARRAY^TM样品上样溶液。394 primer pairs were designed to PCR amplify exons from the genes BRCA1, BRCA2, PTEN, PI3KCA, APC, EGFR, and TP53 (see Table 6 below). The forward primers were appended with the Tag8 sequence, and the reverse primers were appended with the Tag5 sequence. The 394 primers were arranged into 48 sets, each containing an average of approximately 8 primer pairs, at a concentration of 1 μM per primer in 0.05% Tween-20. Sample mixtures were prepared from 48 cell line genomic DNA samples (see Table 7 below) by adding 1 μl of sample (50 ng/ul) to 3 μl of sample premix containing 1 U Roche Faststart HiFi polymerase, 1x buffer, 100 μM dNTPs, 4.5 mM_MgCl2 , 5% DMSO, and 1x ACCESS ARRAY^™ sample loading solution.

ACCESS ARRAY^TM IFC按照ACCESS ARRAY^TM User Guide中的说明运行。将样品混合物上样到ACCESS ARRAY 48.48^TM IFC的样品端口。将各组引物上样到ACCESS ARRAY48.48^TM IFC的入口。在Fluidigm独立热循环仪上利用热循环仪供应的标准PCR方案进行PCR。在PCR后，利用分别的控制器(controller)从ACCESS ARRAY^TM IFC收获产物。然后将一微升的每个产物转移到PCR板并用PCR级水稀释100x。然后制备包含4μl PCR主混合物(1URoche Faststart HiFi聚合酶、1x缓冲液、100μMdNTP、4.5mM MgCl₂、5％DMSO和如以下表8中所述的条形码引物)的3个PCR板。板1包含PE2-CS1/PE1-BC-CS2形式的带有条形码FL001-FL0048的引物对，每个引物具有400nM的浓度。板2包含PE2-CS2/PE1-BC-CS1形式的带有条形码FL001-FL0048的引物对，每个引物以400nM的浓度。板3包含PE2-CS1/PE2-CS2/PE1-BC-CS1/PE1-BC-CS2形式的带有条形码FL0049-FL0096的两对引物。对所有三个板利用以下热循环方案进行15个PCR循环(95℃10min；15x(95℃15s,60℃30s,72℃90s)；72℃3min)。The ACCESS ARRAY^™ IFC was run according to the instructions in the ACCESS ARRAY^™ User Guide. The sample mixture was loaded into the sample port of the ACCESS ARRAY 48.48^™ IFC. Each set of primers was loaded into the inlet of the ACCESS ARRAY 48.48^™ IFC. PCR was performed on a Fluidigm stand-alone thermal cycler using the standard PCR protocol supplied with the thermal cycler. After PCR, products were harvested from the ACCESS ARRAY^™ IFC using separate controllers. One microliter of each product was then transferred to a PCR plate and diluted 100x with PCR-grade water. Three PCR plates were then prepared containing 4 μl of PCR master mix (1U Roche Faststart HiFi polymerase, 1x buffer, 100 μM dNTPs, 4.5 mM MgCl₂ , 5% DMSO, and barcode primers as described in Table 8 below). Plate 1 contained a primer pair with barcodes FL001-FL0048 in the format of PE2-CS1/PE1-BC-CS2, each at a concentration of 400 nM. Plate 2 contained a primer pair with barcodes FL001-FL0048 in the format of PE2-CS2/PE1-BC-CS1, each at a concentration of 400 nM. Plate 3 contained two pairs of primers with barcodes FL0049-FL0096 in the format of PE2-CS1/PE2-CS2/PE1-BC-CS1/PE1-BC-CS2. 15 PCR cycles were performed on all three plates using the following thermal cycling protocol (95°C for 10 min; 15x (95°C for 15 s, 60°C for 30 s, 72°C for 90 s); 72°C for 3 min).

在Agilent 1000 Bioanalyzer芯片上分析来自每个板的每个反应产物，基于来自分析的电泳图测量PCR产物池的浓度(图28)。利用根据从AgilentBioanalyzer获得的浓度调整的体积，将来自每个板的PCR产物汇集到相等浓度。Each reaction product from each plate was analyzed on an Agilent 1000 Bioanalyzer chip and the concentration of the PCR product pool was measured based on the electropherogram from the analysis ( FIG. 28 ). The PCR products from each plate were pooled to equal concentrations using volumes adjusted according to the concentrations obtained from the Agilent Bioanalyzer.

利用AMPure珠(Beckman Coulter)清洁汇集的样品，珠与样品的比例为1：1。Pooled samples were cleaned using AMPure beads (Beckman Coulter) at a 1:1 beads to sample ratio.

扩增子池在Genome Analyzer II(Illumina)的两个分别的泳道上测序。第一泳道利用CS1和CS2引物用于第一读取，和C1rc和CS2rc引物用于索引读取。因为预计CS1和CS2的退火温度比标准Illumina读取1和索引测序引物的低10℃，使用CS1、CS2、CS1rc和CS2rc的LNA(锁核酸)形式以优化在Illumina Cluster Station and Genome Analyzer手册中描述的标准条件下对簇的杂交。The amplicon pool was sequenced in two separate lanes of a Genome Analyzer II (Illumina). The first lane utilized CS1 and CS2 primers for the first read, and C1rc and CS2rc primers for the index read. Because the annealing temperature of CS1 and CS2 was expected to be 10°C lower than that of standard Illumina read 1 and index sequencing primers, LNA (locked nucleic acid) forms of CS1, CS2, CS1rc, and CS2rc were used to optimize hybridization to the cluster under standard conditions described in the Illumina Cluster Station and Genome Analyzer manual.

对于测序，第二泳道使用从在ACCESS ARRAY^TM IFC上扩增期间使用的引物组装的靶特异性正向和反向引物的池(图29)。CS1/CS2rc索引引物用于索引读取。由于其长度增加，靶特异性引物具有比CS1或CS2高的退火温度。这一方法避免了读取通过(readingthrough)PCR产物的不提供信息的靶特异性引物部分。相反，从PCR产物的提供信息的区域获得具有最低误差率的测序信息，在该区域5'和3'读取之间存在最少量的重叠。方法还允许测序误差率最大处(即，PCR产物中部)的更大重叠，和PCR产物尺寸增加30-40bp。For order-checking, the second swimming lane uses the pool (Figure 29) of the target-specific forward and reverse primers assembled from the primers used during the amplification on ACCESS ARRAY^™ IFC. CS1/CS2rc index primer is used for index reading. Due to its length increase, the target-specific primer has an annealing temperature higher than CS1 or CS2. This method avoids reading the target-specific primer part that does not provide information by (reading through) PCR product. On the contrary, the order-checking information with the lowest error rate is obtained from the informative region of PCR product, and there is a minimum overlap between reading in this region 5 ' and 3 '. Method also allows the larger overlap of the maximum place (that is, the middle part of the PCR product) of the order-checking error rate, and the PCR product size increases by 30-40bp.

利用Illumina软件将序列数据多路分用(demultiplexed)并利用aligner ELAND(Illumina)与人类基因组参考序列build hg19比对。示例性的样品的基因EGFR的逐个碱基覆盖显示在图30。Sequence data were demultiplexed using Illumina software and aligned to the human genome reference sequence build hg 19 using the aligner ELAND (Illumina). Base-by-base coverage of the gene EGFR for exemplary samples is shown in FIG30 .

表6用于扩增来自基因BRCA1、BRCA2、PTEN、PI3KCA、APC、EGFR、TP53的外显子的引物Table 6 Primers for amplifying exons from genes BRCA1, BRCA2, PTEN, PI3KCA, APC, EGFR, and TP53

表7细胞系基因组DNA样品Table 7 Cell line genomic DNA samples

样品sampleBCBCK562K562FLD0001FLD0001MOLT-4MOLT-4FLD0002FLD0002CCRF-CEMCCRF-CEMFLD0003FLD0003RPMI-8226RPMI-8226FLD0004FLD0004HL-60(TB)HL-60(TB)FLD0005FLD0005SRSRFLD0006FLD0006SF-268SF-268FLD0007FLD0007SF-295SF-295FLD0008FLD0008SF-539SF-539FLD0009FLD0009SNB-19SNB-19FLD0010FLD0010SNB-75SNB-75FLD0011FLD0011U251U251FLD0012FLD0012BT-549BT-549FLD0013FLD0013HS-578THS-578TFLD0014FLD0014MCF7MCF7FLD0015FLD0015NCI/ADR-RESNCI/ADR-RESFLD0016FLD0016MBA-MB-231/ATCCMBA-MB-231/ATCCFLD0017FLD0017MDA-MB-435MDA-MB-435FLD0018FLD0018T-47DT-47DFLD0019FLD0019COLO 205COLO 205FLD0020FLD0020HCC-2998HCC-2998FLD0021FLD0021HCT-116HCT-116FLD0022FLD0022HCT-15HCT-15FLD0023FLD0023HT-29HT-29FLD0024FLD0024KM12KM12FLD0025FLD0025SW-620SW-620FLD0026FLD0026A549/ATCCA549/ATCCFLD0027FLD0027EKVXEKVXFLD0028FLD0028HOP-62HOP-62FLD0029FLD0029HOP-92HOP-92FLD0030FLD0030NCI-H322MNCI-H322MFLD0031FLD0031NC I-H226NC I-H226FLD0032FLD0032NCI-H23NCI-H23FLD0033FLD0033NCI-H460NCI-H460FLD0034FLD0034NCI-H522NCI-H522FLD0035FLD0035LOX IMVILOX IMVIFLD0036FLD0036M14M14FLD0037FLD0037MALME-3MMALME-3MFLD0038FLD0038SK-MEL-2SK-MEL-2FLD0039FLD0039

样品sampleBCBCSK-MEL-28SK-MEL-28FLD0040FLD0040SK-MEL-5SK-MEL-5FLD0041FLD0041UACC-257UACC-257FLD0042FLD0042UACC-62UACC-62FLD0043FLD0043IGR-OV1IGR-OV1FLD0044FLD0044OVCAR-3OVCAR-3FLD0045FLD0045OVCAR-4OVCAR-4FLD0046FLD0046OVCAR-5OVCAR-5FLD0047FLD0047OVCAR-8OVCAR-8FLD0048FLD0048K562K562FLD0049FLD0049MOLT-4MOLT-4FLD0050FLD0050CCRF-CEMCCRF-CEMFLD0051FLD0051RPMI-8226RPMI-8226FLD0052FLD0052HL-60(TB)HL-60(TB)FLD0053FLD0053SRSRFLD0054FLD0054SF-268SF-268FLD0055FLD0055SF-295SF-295FLD0056FLD0056SF-539SF-539FLD0057FLD0057SNB-19SNB-19FLD0058FLD0058SNB-75SNB-75FLD0059FLD0059U251U251FLD0060FLD0060BT-549BT-549FLD0061FLD0061HS-578THS-578TFLD0062FLD0062MCF7MCF7FLD0063FLD0063NCI/ADR-RESNCI/ADR-RESFLD0064FLD0064MBA-MB-231/ATCCMBA-MB-231/ATCCFLD0065FLD0065MDA-MB-435MDA-MB-435FLD0066FLD0066T-47DT-47DFLD0067FLD0067COLO 205COLO 205FLD0068FLD0068HCC-2998HCC-2998FLD0069FLD0069HCT-116HCT-116FLD0070FLD0070HCT-15HCT-15FLD0071FLD0071HT-29HT-29FLD0072FLD0072KM12KM12FLD0073FLD0073SW-620SW-620FLD0074FLD0074A549/ATCCA549/ATCCFLD0075FLD0075EKVXEKVXFLD0076FLD0076HOP-62HOP-62FLD0077FLD0077HOP-92HOP-92FLD0078FLD0078NCI-H322MNCI-H322MFLD0079FLD0079NCI-H226NCI-H226FLD0080FLD0080NCI-H23NCI-H23FLD0081FLD0081

样品sampleBCBCNCI-H460NCI-H460FLD0082FLD0082NCI-H522NCI-H522FLD0083FLD0083LOX IMVILOX IMVIFLD0084FLD0084M14M14FLD0085FLD0085MALME-3MMALME-3MFLD0086FLD0086SK-MEL-2SK-MEL-2FLD0087FLD0087SK-MEL-28SK-MEL-28FLD0088FLD0088SK-MEL-5SK-MEL-5FLD0089FLD0089UACC-257UACC-257FLD0090FLD0090UACC-62UACC-62FLD0091FLD0091IGR-OV1IGR-OV1FLD0092FLD0092OVCAR-3OVCAR-3FLD0093FLD0093OVCAR-4OVCAR-4FLD0094FLD0094OVCAR-5OVCAR-5FLD0095FLD0095OVCAR-8OVCAR-8FLD0096FLD0096

表8条形码引物Table 8 Barcode primers

实施例11Example 11

利用48.48 ACCESS ARRAY^TM IFC、用于Illumina测序仪的双向DNA测序扩增子加标Bidirectional DNA Sequencing Amplicon Spiking for Illumina Sequencers Using the 48.48 ACCESS ARRAY^™ IFC签-方案2Sign-Option 2

这一实施例提供实施例9中的方案的修改形式。实施例9的引言也适用于这一实施例。This embodiment provides a modification of the approach in embodiment 9. The introduction to embodiment 9 also applies to this embodiment.

制备扩增子Preparation of amplicons

可查阅以下文件用作这一方案的参考：IFC Controller for ACCESSARRAY^TM System User Guide(PN 68000157)；Control Line Fluid LoadingProcedure Quick Reference(PN68000132)；和Agilent DNA 1000 Kit Guide。The following documents may be consulted for reference regarding this protocol: IFC Controller for ACCESSARRAY^™ System User Guide (PN 68000157); Control Line Fluid Loading Procedure Quick Reference (PN 68000132); and Agilent DNA 1000 Kit Guide.

以下试剂用于这一方案并储存在-20℃：FastStart高保真PCR System,dNTPack(Roche,PN 04-738-292-001)；20X ACCESS ARRAY^TM上样试剂(Fluidigm,PN 100-0883)；1XACCESS ARRAY^TM收获溶液(Fluidigm,PN100-1031)；用于Illumina测序仪的ACCESS ARRAY^TM条形码文库-384(双向)(Fluidigm,PN 100-3771)；以通用标签(CS1正向标签、CS2反向标签)加标签的靶特异性引物对，包括50μM CS1-加标签的TS正向引物和50μMCS2-加标签的TS反向引物；和50 ng/μL的模板DNA。(1X ACCESS ARRAY^TM收获溶液(Fluidigm,PN 100-1031)不单独包装出售。它可以ACCESS ARRAY^TM Harvest Pack,PN 100-3155的名称以10个单位购买，或作为48.48 ACCESS ARRAY^TM上样试剂试剂盒,PN 100-1032中的组分购买。)还使用Agilent DNA 1000试剂盒试剂(Agilent,PN 5067-1504)，其储存在4℃。另外，使用PCRCertified Water(Teknova,PN W330)；其储存在室温。The following reagents were used in this protocol and stored at −20°C: FastStart High-Fidelity PCR System, dNTPack (Roche, PN 04-738-292-001); 20× ACCESS ARRAY^™ loading reagent (Fluidigm, PN 100-0883); 1× ACCESS ARRAY^™ harvest solution (Fluidigm, PN 100-1031); ACCESS ARRAY^™ barcoded library for Illumina sequencers - 384 (bidirectional) (Fluidigm, PN 100-3771); target-specific primer pairs tagged with universal tags (CS1 forward tag, CS2 reverse tag), including 50 μM CS1-tagged TS forward primer and 50 μM CS2-tagged TS reverse primer; and 50 ng/μL template DNA. (1X ACCESS ARRAY^™ Harvest Solution (Fluidigm, PN 100-1031) is not sold individually. It can be purchased in 10 units as ACCESS ARRAY^™ Harvest Pack, PN 100-3155, or as a component of the 48.48 ACCESS ARRAY^™ Loading Reagent Kit, PN 100-1032.) Agilent DNA 1000 kit reagents (Agilent, PN 5067-1504) were also used and stored at 4° C. In addition, PCR certified water (Teknova, PN W330) was used; it was stored at room temperature.

在ACCESS ARRAY^TM IFC上的多路PCR按照在ACCESS ARRAY^TM System forIllumina Platform User Guide中的Chapter 6-Multiplex Amplicon Tagging on the48.48 ACCESS ARRAY^TM IFC中详述的说明进行。可选地，按照在ACCESS ARRAY^TM Systemfor Illumina Platform User Guide的附录C中详述的说明，进行48.48 ACCESS ARRAY^TMIFC上的2引物靶特异性PCR以实现无多路的双向扩增子加标签。然后按照以下说明将收获的PCR产物条形码化。Multiplex PCR on the ACCESS ARRAY^™ IFC was performed according to the instructions detailed in Chapter 6 - Multiplex Amplicon Tagging on the 48.48 ACCESS ARRAY^™ IFC in the ACCESS ARRAY^™ System for Illumina Platform User Guide. Alternatively, two-primer target-specific PCR on the 48.48 ACCESS ARRAY^™ IFC was performed according to the^instructions detailed in Appendix C of the ACCESS ARRAY™ System for Illumina Platform User Guide to achieve bidirectional amplicon tagging without multiplexing. The harvested PCR products were then barcoded according to the following instructions.

按照在Fluidigm ACCESS ARRAY^TM System for Illumina Platform User Guide中Chapter 6-Attaching Sequence Tags and Sample Barcodes中详述的说明，在两个96孔板中将PCR产物条形码化用于双向扩增子加标签。收获的PCR产物池的100倍稀释物用作二个(而不是一个)条形码化PCR反应的模板：一个96孔板中一个反应产生的PCR产物A允许测序靶区域的5'末端，第二96孔板中另一个反应产生的PCR产物B允许测序靶区域的3'末端。反应的设置与Fluidigm ACCESS ARRAY^TM System for Illumina Platform User Guide中的"Attaching Sequence Tags and Sample Barcodes"相同。然而，样品预混合物溶液的量加倍以补偿反应数目的增加，且用于Illumina测序仪的ACCESS ARRAY^TM条形码文库-384(双向)(Fluidigm,PN 100-3771)用于制备样品混合物溶液(表9和10)。PCR products were barcoded for bidirectional amplicon tagging in two 96-well plates, following the instructions detailed in Chapter 6 - Attaching Sequence Tags and Sample Barcodes in the Fluidigm ACCESS ARRAY^™ System for Illumina Platform User Guide. A 100-fold dilution of the harvested PCR product pool was used as template for two (not one) barcoding PCR reactions: PCR product A generated from one reaction in one 96-well plate allowed sequencing of the 5' end of the target region, and PCR product B generated from another reaction in a second 96-well plate allowed sequencing of the 3' end of the target region. The reaction setup was identical to the "Attaching Sequence Tags and Sample Barcodes" section in the Fluidigm ACCESS ARRAY^™ System for Illumina Platform User Guide. However, the amount of sample premix solution was doubled to compensate for the increase in the number of reactions, and ACCESS ARRAY^™ Barcode Library-384 (Bidirectional) for Illumina Sequencer (Fluidigm, PN 100-3771) was used to prepare the sample mix solution (Tables 9 and 10).

表9样品混合溶液-PCR产物ATable 9 Sample mixed solution-PCR product A

组分Components体积(μL)Volume (μL)样品预混合物Sample premix15.015.04.04.0稀释的收获的PCR产物池Diluted harvested PCR product pool1.01.0共common20.020.0

表10样品混合溶液-PCR产物BTable 10 Sample mixed solution-PCR product B

组分Components体积(μL)Volume (μL)样品预混合物Sample premix15.015.04.04.0稀释的收获的PCR产物池Diluted harvested PCR product pool1.01.0共common20.020.0

在第二PCR完成后，合并PCR产物A和PCR产物B池，随后测序。Fluidigm ACCESSARRAY^TM System for Illumina Platform User Guide的第8章提供描述PCR后产物文库纯化和定量的方法。使用用于Illumina测序仪的ACCESS ARRAY^TM条形码文库-384(双向)(Fluidigm,PN 100-3771)来产生用于测序的双向扩增子是关键的。After the second PCR is complete, PCR product A and PCR product B pools are combined and subsequently sequenced. Chapter 8 of the Fluidigm ACCESSARRAY^™ System for Illumina Platform User Guide provides methods describing post-PCR product library purification and quantification. It is critical to use the ACCESS ARRAY^™ Barcode Library-384 (Bidirectional) (Fluidigm, PN 100-3771) for Illumina sequencers to generate bidirectional amplicons for sequencing.

利用Fluidigm FL1和FL2测序引物的测序工作流Sequencing workflow using Fluidigm FL1 and FL2 sequencing primers

以下说明预期与Illumina TruSeq测序试剂在Illumina GAII和HiSeq系统上一起使用。Fluidigm测序试剂FL1和FL2分别包含CS1和CS2测序和索引引物的等摩尔混合物。FL1是定制的测序引物，包含各50μM的CS1和CS2引物。FL2是定制的索引引物，包含各50μM的CS1rc和CS2rc引物。对于单读取测序，对读取1和索引引物制备试剂。对于末端配对的测序，对读取1、索引和读取2引物制备试剂。The following instructions are intended for use with Illumina TruSeq sequencing reagents on the Illumina GAII and HiSeq systems. Fluidigm sequencing reagents FL1 and FL2 contain equimolar mixtures of CS1 and CS2 sequencing and indexing primers, respectively. FL1 is a custom sequencing primer containing 50 μM each of CS1 and CS2 primers. FL2 is a custom indexing primer containing 50 μM each of CS1rc and CS2rc primers. For single-read sequencing, prepare reagents for Read 1 and Index primers. For paired-end sequencing, prepare reagents for Read 1, Index, and Read 2 primers.

试验Fluidigm测序引物和TruSeq测序引物之间相互干扰的PCR实验的结果显示在图32和33。The results of PCR experiments testing the mutual interference between Fluidigm sequencing primers and TruSeq sequencing primers are shown in Figures 32 and 33.

可查阅以下文件作为测序的参考：Illumina cBot^TM User Guide；IlluminaGenome Analyzer II^TM User Guide；和Illumina HiSeq^TM User Guide。应参考IlluminaGenome Analyzer II User Guide或Illumina HiSeq User Guide对于如何进行测序运行的说明。还可联系Illumina的技术支持。The following documents are available as references for sequencing: Illumina cBot^™ User Guide; Illumina Genome Analyzer II^™ User Guide; and Illumina HiSeq^™ User Guide. The Illumina Genome Analyzer II User Guide or Illumina HiSeq User Guide should be referenced for instructions on how to perform a sequencing run. You may also contact Illumina technical support.

制备用于在Illumina GAII和HiSeq测序系统上测序的试剂Prepare reagents for sequencing on the Illumina GAII and HiSeq sequencing systems

读取1测序引物HT1/FL1通过在无DNA酶、RNA酶的1.5mL微量离心管中首先用杂交缓冲液(HT1)稀释FL1母液到终浓度500nM来制备(表11)。涡旋试管至少20秒，并离心30秒以旋降(spin down)所有组分。以下说明概述了用于读取1的HT1/FL1测序引物混合物的制备(每mL)。每泳道使用大约300μL，利用cBot Custom Primers Reagent Stage。管条中的定制引物方向与GAII或HiSeq流动小室的泳道对齐。Read 1 sequencing primer HT1/FL1 was prepared by first diluting the FL1 stock solution with hybridization buffer (HT1) to a final concentration of 500 nM in a DNase- and RNase-free 1.5 mL microcentrifuge tube (Table 11). The tube was vortexed for at least 20 seconds and centrifuged for 30 seconds to spin down all components. The following instructions outline the preparation of the HT1/FL1 sequencing primer mixture for read 1 (per mL). Approximately 300 μL was used per lane using the cBot Custom Primers Reagent Stage. The custom primer orientation in the tube strip was aligned with the lanes of the GAII or HiSeq flow cell.

表11制备HT1/FL1(每mL)的说明Table 11 Instructions for preparing HT1/FL1 (per mL)

试剂Reagents体积(μL)Volume (μL)HT1缓冲液HT1 buffer990μL990μLFL1母液(来自附录E)FL1 stock solution (from Appendix E)10μL10 μL共common1.0mL1.0mL

索引引物HT1/FL2通过在无DNA酶、RNA酶的1.5mL微量离心管中首先用杂交缓冲液(HT1)稀释FL2母液到终浓度500nM来制备(表12)。涡旋试管至少20秒，并离心30秒以旋降所有组分。以下说明概述了用于索引读取的HT1/FL2索引引物混合物的制备。大约3mL索引测序引物混合物(HP8)用于索引读取。1.5mL TruSeq试剂HP8代替1.5mL HT1/FL2。Index primers HT1/FL2 were prepared by first diluting the FL2 stock solution to a final concentration of 500 nM with hybridization buffer (HT1) in a DNase- and RNase-free 1.5 mL microcentrifuge tube (Table 12). The tube was vortexed for at least 20 seconds and centrifuged for 30 seconds to spin down all components. The following instructions outline the preparation of the HT1/FL2 index primer mix for index reads. Approximately 3 mL of index sequencing primer mix (HP8) was used for index reads. 1.5 mL of TruSeq reagent HP8 was used in place of 1.5 mL of HT1/FL2.

表12制备HT1/FL2的说明Table 12 Description of the preparation of HT1/FL2

试剂Reagents体积volumeH川缓冲液H-Kawabuffered saline1,485μL1,485 μL厂L]母液@来自附录EPlant L] mother liquor @ from Appendix E15μL15 μL共common1.5mL1.5mL

读取2测序引物HT1/FL1(用于末端配对的测序)通过在无DNA酶、RNA酶的1.5mL微量离心管中首先用杂交缓冲液(HT1)稀释FL1母液到终浓度500nM来制备(表13)。涡旋试管至少20秒，并离心30秒以旋降所有组分。以下说明概述了用于读取2的HT1/FL1测序引物混合物的制备。大约3.2mL读取2测序引物(HP7)用于读取2。1.6mL TruSeq试剂HP7代替1.6mLHT1/FL1。Read 2 sequencing primers HT1/FL1 (for paired-end sequencing) were prepared by first diluting the FL1 stock solution with hybridization buffer (HT1) to a final concentration of 500 nM in a DNase- and RNase-free 1.5 mL microcentrifuge tube (Table 13). The tube was vortexed for at least 20 seconds and centrifuged for 30 seconds to spin down all components. The following instructions outline the preparation of the HT1/FL1 sequencing primer mix for read 2. Approximately 3.2 mL of read 2 sequencing primer (HP7) was used for read 2. 1.6 mL of TruSeq reagent HP7 replaced 1.6 mL of HT1/FL1.

表13制备读取2测序引物HT1/FL1的说明Table 13 Instructions for preparing read 2 sequencing primers HT1/FL1

试剂Reagents体积volumeHT1缓冲液HT1 buffer1,584μL1,584μLFL1母液(来自附录E)FL1 stock solution (from Appendix E)16μL16μL共common1.6mL1.6mL

进行测序运行Perform sequencing runs

Illumina Genome Analyzer II或HiSeq用户指南提供如何进行测序运行的说明。可选地，可联系Illumina的技术支持。The Illumina Genome Analyzer II or HiSeq User Guide provides instructions on how to perform a sequencing run. Alternatively, you can contact Illumina Technical Support.

对于索引读取，1.5mL TruSeq试剂HP8替换为1.5mL索引引物HT1/FL2用于GAII和HiSeq测序运行。用于Illumina的ACCESS ARRAY^TM条形码文库中使用的条形码序列设计为使得即使存在测序误差时仍可区分它们。随着平行运行更多样品，区分条形码序列所需的索引读取的长度明显增加。索引读取的建议描述在表14。For index reads, 1.5 mL of TruSeq reagent HP8 was replaced with 1.5 mL of index primers HT1/FL2 for both GAII and HiSeq sequencing runs. The barcode sequences used in the ACCESS ARRAY^™ barcode library for Illumina were designed to allow for differentiation even in the presence of sequencing errors. As more samples are run in parallel, the length of the index reads required to differentiate between barcode sequences increases significantly. Recommendations for index reads are described in Table 14.

表14索引读取建议Table 14 Index read recommendations

每泳道的样品数目Number of samples per lane1-3841-384385-1920385-1920索引读取的长度Length of index reads8个碱基8 bases10个碱基10 bases

制备测序运行时，按照表14中的指导调整索引读取的长度。确保上样到测序仪的测序试剂的体积足够用于索引循环。对于如何实现这些改变的详细说明，查阅IlluminaSequencer User Guide，或联系Illumina的技术支持。When preparing for a sequencing run, adjust the length of the index reads according to the guidelines in Table 14. Ensure that the volume of sequencing reagents loaded onto the sequencer is sufficient for the index cycle. For detailed instructions on how to make these changes, consult the Illumina Sequencer User Guide or contact Illumina Technical Support.

Claims (41)

Translated fromChinese

1.一种扩增靶核酸的方法，所述方法包括：1. A method for amplifying a target nucleic acid, the method comprising:利用以下扩增靶核酸：Target nucleic acid was amplified using:内侧引物组，其中该组包含：An inner primer set, wherein the set comprises:内侧、正向引物，包含靶特异性部分和第一引物结合位点；an inner, forward primer containing a target-specific portion and a first primer binding site;内侧、反向引物，包含靶特异性部分和第二引物结合位点，其中所述第一和第二引物结合位点是不同的并且是DNA测序引物的结合位点；an inner, reverse primer comprising a target-specific portion and a second primer binding site, wherein the first and second primer binding sites are different and are binding sites for a DNA sequencing primer;第一外侧引物组，其中该组包含：A first outer primer set, wherein the set comprises:第一外侧、正向引物，包含对所述第一引物结合位点特异性的部分；和a first outer, forward primer comprising a portion specific for said first primer binding site; and第一外侧、反向引物，包含条形码核苷酸序列和对所述第二引物结合位点特异性的部分；a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for the second primer binding site;第二外侧引物组，其中该组包含：a second outer primer set, wherein the set comprises:第二外侧、正向引物，包含条形码核苷酸序列和对所述第一引物结合位点特异性的部分；和a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and第二外侧、反向引物，包含对所述第二引物结合位点特异性的部分；a second outer, reverse primer comprising a portion specific for the second primer binding site;以产生两种靶扩增子，其中：to produce two target amplicons, where:第一靶扩增子包含5'-第一引物结合位点-靶核苷酸序列-第二引物结合位点-条形码核苷酸序列-3'；和The first target amplicon comprises 5'-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-3'; and第二靶扩增子包含5'-条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-3'；The second target amplicon comprises 5'-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-3';其中所述方法包括扩增多个核酸靶并产生具有共同的第一引物结合位点和共同的第二引物结合位点的靶扩增子，其中至少一些靶扩增子具有不同的条形码核苷酸序列。The method comprises amplifying a plurality of nucleic acid targets and generating target amplicons having a common first primer binding site and a common second primer binding site, wherein at least some of the target amplicons have different barcode nucleotide sequences.2.如权利要求1所述的方法，其中从给定的靶核酸产生的每个靶扩增子中的所述条形码核苷酸序列是相同的，且其中每个靶扩增子包含仅一个条形码核苷酸序列。2. The method of claim 1, wherein the barcode nucleotide sequence in each target amplicon generated from a given target nucleic acid is identical, and wherein each target amplicon comprises only one barcode nucleotide sequence.3.如权利要求1-2任一项所述的方法，其中所述外侧引物各自另外包含另外的核苷酸序列，其中：3. The method of any one of claims 1 to 2, wherein each of the outer primers further comprises an additional nucleotide sequence, wherein:所述第一外侧、正向引物包含第一另外的核苷酸序列，且所述第一外侧、反向引物包含第二另外的核苷酸序列；和The first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and所述第二外侧、正向引物包含所述第二另外的核苷酸序列，且所述第二外侧、反向引物包含所述第一另外的核苷酸序列；和said second outer, forward primer comprises said second additional nucleotide sequence, and said second outer, reverse primer comprises said first additional nucleotide sequence; and所述第一和第二另外的核苷酸序列是不同的；和the first and second additional nucleotide sequences are different; and所述扩增产生两种靶扩增子，其中：The amplification produces two target amplicons, wherein:第一靶扩增子包含5'-第一另外的核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-条形码核苷酸序列-第二另外的核苷酸序列-3'；和The first target amplicon comprises 5′-first additional nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-second additional nucleotide sequence-3′; and第二靶扩增子包含5'-第二另外的核苷酸序列-条形码核苷酸序列-第一引物结合位点-靶核苷酸序列-第二引物结合位点-第一另外的核苷酸序列3'。The second target amplicon comprises 5′-second additional nucleotide sequence-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first additional nucleotide sequence 3′.4.如权利要求3所述的方法，其中所述第一或第二另外的核苷酸序列包含引物结合位点。The method of claim 3 , wherein the first or second additional nucleotide sequence comprises a primer binding site.5.如权利要求4所述的方法，其中所述第一和第二另外的核苷酸序列包含引物结合位点。5. The method of claim 4, wherein the first and second additional nucleotide sequences comprise primer binding sites.6.如权利要求3所述的方法，其中所述第一外侧引物组包含为SEQ ID NO:9的PE1-CS1和为SEQ ID NO:10的PE2-BC-CS2，且所述第二外侧引物组包含为SEQ ID NO:11的PE1-CS2和为SEQ ID NO:12的PE2-BC-CS1。6. The method of claim 3, wherein the first outer primer set comprises PE1-CS1 of SEQ ID NO: 9 and PE2-BC-CS2 of SEQ ID NO: 10, and the second outer primer set comprises PE1-CS2 of SEQ ID NO: 11 and PE2-BC-CS1 of SEQ ID NO: 12.7.如权利要求1所述的方法，其中所述扩增在单个扩增反应中进行。7. The method of claim 1, wherein the amplification is performed in a single amplification reaction.8.如权利要求1所述的方法，其中所述扩增包括在第一扩增反应中采用所述内侧引物和在第二扩增反应中采用所述外侧引物，其中所述第二扩增反应不同于所述第一扩增反应。8. The method of claim 1, wherein the amplifying comprises employing the inner primers in a first amplification reaction and the outer primers in a second amplification reaction, wherein the second amplification reaction is different from the first amplification reaction.9.如权利要求8所述的方法，其中所述第二扩增反应包括两个分别的扩增反应，其中一个扩增反应采用所述第一外侧引物组且另一个扩增反应采用所述第二外侧引物组。9. The method of claim 8, wherein the second amplification reaction comprises two separate amplification reactions, one amplification reaction employing the first outer primer set and the other amplification reaction employing the second outer primer set.10.如权利要求9所述的方法，其中汇集在所述两个分别的扩增反应中产生的靶扩增子。10. The method of claim 9, wherein the target amplicons produced in the two separate amplification reactions are pooled.11.如权利要求1所述的方法，其中所述多个靶核酸选自以下组成的组：基因组DNA、cDNA、片段化的DNA、从RNA逆转录的DNA、DNA文库、和从细胞、体液或组织样品提取或扩增的核酸。11. The method of claim 1, wherein the plurality of target nucleic acids are selected from the group consisting of genomic DNA, cDNA, fragmented DNA, DNA reverse transcribed from RNA, a DNA library, and nucleic acids extracted or amplified from cells, body fluids, or tissue samples.12.如权利要求11所述的方法，其中所述多个靶核酸是从福尔马林固定的、石蜡包埋的组织样品扩增的。12. The method of claim 11, wherein the plurality of target nucleic acids are amplified from a formalin-fixed, paraffin-embedded tissue sample.13.如权利要求1所述的方法，其中所述方法另外包括测序所述靶扩增子。13. The method of claim 1, wherein the method further comprises sequencing the target amplicon.14.如权利要求1所述的方法，其中所述方法包括利用结合所述第一和第二另外的核苷酸序列的引物扩增所述靶扩增子以产生用于DNA测序的模板。14. The method of claim 1, wherein the method comprises amplifying the target amplicon using primers that bind to the first and second additional nucleotide sequences to generate a template for DNA sequencing.15.如权利要求14所述的方法，其中结合所述第一和第二另外的核苷酸序列的引物之一或二者被固定在基质上。15. The method of claim 14, wherein one or both of the primers that bind to the first and second additional nucleotide sequences are immobilized on a substrate.16.如权利要求1所述的方法，其中所述扩增通过等温核酸扩增进行。16. The method of claim 1, wherein the amplifying is performed by isothermal nucleic acid amplification.17.如权利要求14所述的方法，其中所述方法包括利用所述模板和结合所述第一和第二引物结合位点的靶测序引物进行DNA测序，其中所述靶测序引物引发所述靶核苷酸序列的测序。17. The method of claim 14, wherein the method comprises performing DNA sequencing using the template and a target sequencing primer that binds to the first and second primer binding sites, wherein the target sequencing primer primes sequencing of the target nucleotide sequence.18.如权利要求17所述的方法，其中所述靶测序引物以大致上相等的量存在以产生来自每个靶核苷酸序列的5'和3'DNA序列信息。18. The method of claim 17, wherein the target sequencing primers are present in substantially equal amounts to generate 5' and 3' DNA sequence information from each target nucleotide sequence.19.如权利要求14所述的方法，其中所述方法包括利用所述模板和结合所述第一和第二引物结合位点并引发所述条形码核苷酸序列的测序的引物进行DNA测序。19. The method of claim 14, wherein the method comprises performing DNA sequencing using the template and primers that bind to the first and second primer binding sites and prime the sequencing of the barcode nucleotide sequence.20.如权利要求19所述的方法，其中结合所述第一和第二引物结合位点并引发所述条形码核苷酸序列的测序的所述引物以大致上相等的量存在。20. The method of claim 19, wherein the primers that bind to the first and second primer binding sites and prime the sequencing of the barcode nucleotide sequence are present in substantially equal amounts.21.如权利要求17所述的方法，其中所述方法包括利用所述模板和结合所述第一和第二引物结合位点并引发所述条形码核苷酸序列的测序的引物进行DNA测序，其中所述引物是引发所述靶核苷酸序列的测序的引物的反向互补物。21. The method of claim 17, wherein the method comprises performing DNA sequencing using the template and primers that bind to the first and second primer binding sites and prime the sequencing of the barcode nucleotide sequence, wherein the primers are the reverse complement of the primers that prime the sequencing of the target nucleotide sequence.22.如权利要求21所述的方法，其中用以引发所述靶核苷酸序列和条形码核苷酸序列的测序的所述引物包含为SEQ ID NO:13的CS1、为SEQ ID NO:14的CS2、为SEQ ID NO:15的CS1rc和为SEQ ID NO:16的CS2rc。22. The method of claim 21, wherein the primers used to prime the sequencing of the target nucleotide sequence and the barcode nucleotide sequence comprise CS1 of SEQ ID NO: 13, CS2 of SEQ ID NO: 14, CS1rc of SEQ ID NO: 15, and CS2rc of SEQ ID NO: 16.23.如权利要求1所述的方法，其中选择所述条形码核苷酸序列以避免大致上退火到所述靶核酸。23. The method of claim 1, wherein the barcode nucleotide sequence is selected to avoid substantially annealing to the target nucleic acid.24.如权利要求1所述的方法，其中所述条形码核苷酸序列辨识特定样品。24. The method of claim 1, wherein the barcode nucleotide sequence identifies a specific sample.25.如权利要求1所述的方法，其中所述靶扩增子的平均长度小于200个碱基。25. The method of claim 1, wherein the average length of the target amplicons is less than 200 bases.26.如权利要求1所述的方法，其中所述第一扩增反应在约1皮升至约50纳升的范围的体积中进行。26. The method of claim 1, wherein the first amplification reaction is performed in a volume ranging from about 1 picoliter to about 50 nanoliters.27.如权利要求1所述的方法，其中所述第一扩增反应在约5皮升至约25纳升的范围的体积中进行。27. The method of claim 1, wherein the first amplification reaction is performed in a volume ranging from about 5 picoliters to about 25 nanoliters.28.如权利要求1所述的方法，其中所述第一扩增反应在扩增之前在微流体装置的分别的区室中形成或被分配到微流体装置的分别的区室中。28. The method of claim 1, wherein the first amplification reaction is formed in or partitioned into separate compartments of a microfluidic device prior to amplification.29.如权利要求28所述的方法，其中所述微流体装置至少部分地从弹性体材料制造。29. The method of claim 28, wherein the microfluidic device is fabricated at least in part from an elastomeric material.30.如权利要求1所述的方法，其中所述第一扩增反应在液滴中进行。30. The method of claim 1, wherein the first amplification reaction is performed in a droplet.31.如权利要求30所述的方法，其中多个第一扩增反应在乳液中的液滴中进行。31. The method of claim 30, wherein the plurality of first amplification reactions are performed in droplets in an emulsion.32.一种用于扩增靶核酸以产生权利要求1中定义的所述两种靶扩增子的试剂盒，所述试剂盒包含：32. A kit for amplifying a target nucleic acid to produce the two target amplicons defined in claim 1, the kit comprising:第一外侧引物组，其中该组包含：A first outer primer set, wherein the set comprises:第一外侧、正向引物，包含对第一引物结合位点特异性的部分；和a first outer, forward primer comprising a portion specific for the first primer binding site; and第一外侧、反向引物，包含条形码核苷酸序列和对第二引物结合位点特异性的部分，其中所述第一和第二引物结合位点是不同的并且是DNA测序引物的结合位点；a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for a second primer binding site, wherein the first and second primer binding sites are different and are binding sites for a DNA sequencing primer;第二外侧引物组，其中该组包含：a second outer primer set, wherein the set comprises:第二外侧、正向引物，包含条形码核苷酸序列和对所述第一引物结合位点特异性的部分；和a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and第二外侧、反向引物，包含对所述第二引物结合位点特异性的部分。A second outer, reverse primer comprises a portion specific for the second primer binding site.33.如权利要求32所述的试剂盒，其中所述外侧引物各自另外包含另外的核苷酸序列，其中：33. The kit of claim 32, wherein each of the outer primers further comprises an additional nucleotide sequence, wherein:所述第一外侧、正向引物包含第一另外的核苷酸序列，且所述第一外侧、反向引物包含第二另外的核苷酸序列；和The first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and所述第二外侧、正向引物包含所述第二另外的核苷酸序列，且所述第二外侧、反向引物包含所述第一另外的核苷酸序列；且所述第一和第二另外的核苷酸序列是不同的。The second outer, forward primer comprises the second additional nucleotide sequence, and the second outer, reverse primer comprises the first additional nucleotide sequence; and the first and second additional nucleotide sequences are different.34.如权利要求33所述的试剂盒，其中所述第一外侧引物组包含为SEQ ID NO:9的PE1-CS1和为SEQ ID NO:10的PE2-BC-CS2，且所述第二外侧引物组包含为SEQ ID NO:11的PE1-CS2和为SEQ ID NO:12的PE2-BC-CS1。34. The kit of claim 33, wherein the first outer primer set comprises PE1-CS1 of SEQ ID NO: 9 and PE2-BC-CS2 of SEQ ID NO: 10, and the second outer primer set comprises PE1-CS2 of SEQ ID NO: 11 and PE2-BC-CS1 of SEQ ID NO: 12.35.如权利要求32所述的试剂盒，另外包含：35. The kit of claim 32, further comprising:内侧引物组，其中该组包含：An inner primer set, wherein the set comprises:内侧、正向引物，包含靶特异性部分和所述第一引物结合位点；和an inner, forward primer comprising a target-specific portion and said first primer binding site; and内侧、反向引物，包含靶特异性部分和所述第二引物结合位点。The inner, reverse primer comprises a target-specific portion and the second primer binding site.36.如权利要求35所述的试剂盒，包含各自对不同的靶核酸特异性的多个内侧引物组。36. The kit of claim 35, comprising a plurality of inner primer sets each specific for a different target nucleic acid.37.如权利要求32所述的试剂盒，另外包含结合所述第一和第二引物结合位点并引发所述靶核苷酸序列的测序的DNA测序引物。37. The kit of claim 32, further comprising DNA sequencing primers that bind to the first and second primer binding sites and prime the sequencing of the target nucleotide sequence.38.如权利要求32所述的试剂盒，另外包含结合所述第一和第二引物结合位点并引发所述条形码核苷酸序列的测序的DNA测序引物。38. The kit of claim 32, further comprising DNA sequencing primers that bind to the first and second primer binding sites and prime the sequencing of the barcode nucleotide sequence.39.如权利要求38所述的试剂盒，其中结合所述第一和第二引物结合位点并引发所述条形码核苷酸序列的测序的引物是引发所述靶核苷酸序列的测序的引物的反向互补物。39. The kit of claim 38, wherein the primers that bind to the first and second primer binding sites and prime the sequencing of the barcode nucleotide sequence are the reverse complements of the primers that prime the sequencing of the target nucleotide sequence.40.如权利要求39所述的试剂盒，其中用以引发所述靶核苷酸序列和条形码核苷酸序列的测序的引物包含为SEQ ID NO:13的CS1、为SEQ ID NO:14的CS2、为SEQ ID NO:15的CS1rc和为SEQ ID NO:16的CS2rc。40. The kit of claim 39, wherein the primers for priming the sequencing of the target nucleotide sequence and the barcode nucleotide sequence comprise CS1 of SEQ ID NO: 13, CS2 of SEQ ID NO: 14, CS1rc of SEQ ID NO: 15, and CS2rc of SEQ ID NO: 16.41.如权利要求32所述的试剂盒，其中所述试剂盒另外包含矩阵型微流体装置。41. The kit of claim 32, wherein the kit further comprises a matrix-type microfluidic device.