技术领域technical field
本发明涉及一种核酸物质扩增方法，特别涉及一种能够减小微量核酸物质整体扩增时产生偏倚的扩增方法。The invention relates to a method for amplifying nucleic acid substances, in particular to an amplification method capable of reducing the bias in the overall amplification of trace nucleic acid substances.
背景技术Background technique
随着生命科学研究的不断深入，单细胞水平的分析越来越重要。一方面，由于细胞之间的异质性，一些大量细胞的平均数据并不能真正代表细胞水平上发生的变化，而且往往会掩盖单细胞水平的信号，例如只存在于少数细胞(如早期癌细胞)中的突变等；另一方面，在一些原材料非常稀有的实验中，例如胚胎早期细胞、循环肿瘤细胞和穿刺得到的样品等，很难获得足够进行测序或其他分析的大量细胞。With the deepening of life science research, analysis at the single-cell level is becoming more and more important. On the one hand, due to the heterogeneity among cells, some averaged data of a large number of cells cannot really represent the changes occurring at the cellular level, and tend to mask signals at the single-cell level, such as those present in only a few cells (such as early cancer cells). ) mutations, etc.; on the other hand, in some experiments where raw materials are very rare, such as early embryonic cells, circulating tumor cells, and samples obtained by puncture, it is difficult to obtain a large number of cells enough for sequencing or other analysis.
为了对单个细胞进行测序等分析，必须首先利用包括PCR、MDA、RCA在内的整体扩增技术生成大量的DNA拷贝。然而这些技术存在的一个缺点是：进行整体扩增时，基因组的某些部分相比另一些会生成更大量的拷贝，这会导致基因组中生成拷贝数较少的区域被淹没，从而无法被检测到。这一问题被称作扩增偏倚。扩增偏倚在现有的全基因组扩增方法中广泛存在，并成为重要的不足之处，原因在于扩增偏倚会导致最终产物无法代表片段的起始相对含量，造成错误的染色体倍型和基因拷贝数估计，并且会造成一些片段由于扩增倍数过少而无法在产物中进行检测，使得单细胞分析无法准确进行。此外，在单分子扩增中，由于起始核酸的含量过少，微量的周围环境中的核酸分子或者操作者带入的核酸分子都很容易混入扩增体系中造成污染。In order to perform analysis such as sequencing on a single cell, a large number of DNA copies must first be generated using overall amplification techniques including PCR, MDA, and RCA. However, one disadvantage of these techniques is that when performing global amplification, some parts of the genome will generate a greater number of copies than others, which will cause regions of the genome that generate fewer copies to be overwhelmed and thus cannot be detected arrive. This problem is known as amplification bias. Amplification bias is widespread in existing whole-genome amplification methods and has become an important shortcoming because amplification bias can cause the final product to be unable to represent the initial relative content of the fragment, resulting in wrong chromosome ploidy and gene The copy number is estimated, and some fragments cannot be detected in the product due to too little amplification factor, making single-cell analysis inaccurate. In addition, in single-molecule amplification, due to the low content of the starting nucleic acid, a small amount of nucleic acid molecules in the surrounding environment or nucleic acid molecules brought in by the operator are easily mixed into the amplification system to cause pollution.
发明内容Contents of the invention
针对上述问题，本发明提供了一种能够减小微量核酸物质整体扩增时产生偏倚的扩增方法，其特征在于，将所述核酸物质随机分散到若干互不连通的独立反应体系中后再进行扩增，扩增使用的引物序列中包含3-20个随机碱基，随机碱基随机选自A、G、C、T中的二个、三个或四个。In view of the above problems, the present invention provides an amplification method capable of reducing bias in the overall amplification of trace nucleic acid substances, which is characterized in that the nucleic acid substances are randomly dispersed into several independent reaction systems that are not connected to each other For amplification, the primer sequence used for amplification contains 3-20 random bases, and the random bases are randomly selected from two, three or four of A, G, C, and T.
在传统的大体系(通常指1μL以上，可以直接用移液枪进行操作的体系)单细胞扩增中，单细胞中每个核酸分子或片段在全基因组扩增等同于单分子的扩增，单分子扩增反应的是否启动与何时启动是一个随机过程。一旦一个分子的扩增被随机“启动”了，该分子在短时间内就能够获得高于其他未启动分子数倍的拷贝，而这个数目上的优势则进一步增加了其继续被“启动”的概率，并造成本该属于其他分子的扩增原料被拷贝数较大分子的扩增反应所占用，使得最终产物中某些分子的拷贝数远远大于其他分子，产物中核酸分子拷贝数的比例不能真实反应原料中相同分子的拷贝数比例，这就造成了扩增偏倚。而在本发明的方法中，将待扩增的分子随机分散到若干独立反应体系中，使得每个反应体系中的分子数符合泊松分布，只有0个至50个分子，甚至0-20个，或0-10个以下分子。即便某个分子率先“启动”并获得了较多的拷贝，受其影响的也仅是处于同一反应体系的其他分子，而其他反应体系的分子仍能获得扩增机会。此外，虽然每个反应体系中的单分子扩增的“启动”时间有先有后，但由于每个反应体系中用于扩增的原料是有限的，率先“启动”且扩增速度较快的分子的扩增速度在一段时间后会趋于平稳。当扩增时间足够长，使得反应体系的反应都趋于平稳时同时停止所有的反应体系中的反应，此时理论上所有的分子扩增的倍数应是相同的，从而达到均匀扩增的目的。同时，由于将待扩增的分子分散到了若干个反应体系中，污染物对整体反应的影响就相应的减少了。In the traditional large-scale system (usually refers to the system above 1 μL, which can be directly operated with a pipette gun) single-cell amplification, the whole-genome amplification of each nucleic acid molecule or fragment in a single cell is equivalent to the amplification of a single molecule. Whether and when to start the single molecule amplification reaction is a random process. Once the amplification of a molecule is randomly "started", the molecule can obtain several times more copies than other unstarted molecules in a short period of time, and this number advantage further increases its chances of continuing to be "started". Probability, and cause the amplification raw materials that should belong to other molecules to be occupied by the amplification reaction of larger copy number molecules, so that the copy number of some molecules in the final product is much larger than that of other molecules, the ratio of the copy number of nucleic acid molecules in the product The copy number ratio of the same molecule in the starting material cannot be truly reflected, which causes amplification bias. In the method of the present invention, the molecules to be amplified are randomly dispersed into several independent reaction systems, so that the number of molecules in each reaction system conforms to the Poisson distribution, only 0 to 50 molecules, or even 0-20 , or 0-10 molecules below. Even if a certain molecule "starts" first and obtains more copies, only other molecules in the same reaction system are affected by it, and molecules in other reaction systems still have the opportunity to amplify. In addition, although the "start-up" time of single-molecule amplification in each reaction system is different, because the materials used for amplification in each reaction system are limited, the first "start" and the amplification speed are faster. The rate of amplification of molecules will level off after a period of time. When the amplification time is long enough to make the reactions of the reaction system tend to be stable, the reactions in all the reaction systems are stopped at the same time. At this time, the amplification factor of all molecules should be the same in theory, so as to achieve the purpose of uniform amplification. . At the same time, since the molecules to be amplified are dispersed into several reaction systems, the impact of pollutants on the overall reaction is correspondingly reduced.
进一步的，本发明中扩增使用的引物可以为由3-20个随机碱基组成的序列，随机碱基随机选自A、G、C、T中的二个、三个或四个。本发明的核酸物质的序列可以为未知的。传统的PCR方法中要求被扩增片段至少具有一部分已知序列，再根据这段已知序列设计能够结合在其上的一个或者多个特定序列作为引物。而本发明的方法使用由随机碱基组成的序列作为“随机引物”，由于其随机性可以拥有大量的组合，因此该引物可以和相应长度的未知序列的未知片段进行碱基配对，从而作为引物扩增出未知序列的任意部分，达到未知序列的整体扩增效果，从而允许对未知序列进行全基因组的扩增、测序。根据实验需要，引物中可以包含非自然或者修饰过的碱基，以及与碱基作用的其他物质，例如锁核酸、5′-硝基吲哚、硫代磷酸寡核苷酸等。Further, the primer used for amplification in the present invention may be a sequence consisting of 3-20 random bases, and the random bases are randomly selected from two, three or four of A, G, C, and T. The sequence of the nucleic acid material of the invention may not be known. The traditional PCR method requires that the amplified fragment has at least a part of known sequence, and then design one or more specific sequences that can bind to it as primers according to this known sequence. However, the method of the present invention uses a sequence composed of random bases as a "random primer", which can have a large number of combinations due to its randomness, so the primer can perform base pairing with an unknown fragment of an unknown sequence of a corresponding length, thereby serving as a primer Amplify any part of the unknown sequence to achieve the overall amplification effect of the unknown sequence, thus allowing the amplification and sequencing of the entire genome of the unknown sequence. According to experimental needs, the primers may contain unnatural or modified bases, and other substances that interact with bases, such as locked nucleic acids, 5'-nitroindole, phosphorothioate oligonucleotides, etc.
进一步的，本发明的独立反应体系的体积为0.5fL-100nL，优选1fL-10nL，更优选10fL-1nL。分散至若干独立反应体系中的若干，是指2个以上，优选2-1020个，更优选2-108个。独立反应体系可以由油、水、水凝胶、聚二甲基硅氧烷、玻璃、塑料、硅片、空气中任意不互溶的两相分散而成。两相中的一相能够溶解扩增反应的反应物，称其为分散相，另一相与该相不互溶，称为连续相或分隔相。分散的方法可以是超声分散或使用液体操作系统进行分散。液体操作系统可以是微流控芯片。使用微流芯片产生互不连通的分散相微液滴的方法有多相流法、电润湿法、热毛细管法、介电电泳法等。多相流法的原理是，通过对流体微通道结构的独特设计以及对流体流速的控制，利用液流间的剪切力、黏力和表面张力的相互作用，使分散相流体在微通道局部产生速度梯度，从而被拆分生成微液滴，产生的微液滴均匀地分布在与分散相不互溶的连续相中，形成单分散系统。为了减小表面张力，生成稳定的微液滴，还可以向液流中加入表面活性剂。微流芯片的管道可以是T型，Y型，或十字交叉型。通过调整连续相的流速和连续相与分散相的流比可控制微液滴的形成速度和所形成的微液滴的大小，从而控制独立反应体系的大小和每个反应体系中核酸分子的数量。每个反应体系中核酸物质的含量为0个至50个分子，优选为0-20个分子，更优选为0-10个分子。当体系较大时可以产生更多的产物，核酸分子数量越少，在单个反应室内的相互影响就会越小，可以达到更均匀的扩增Furthermore, the volume of the independent reaction system of the present invention is 0.5fL-100nL, preferably 1fL-10nL, more preferably 10fL-1nL. Several dispersed in several independent reaction systems means 2 or more, preferably 2-1020 , more preferably 2-108 . The independent reaction system can be formed by dispersing any immiscible two-phase in oil, water, hydrogel, polydimethylsiloxane, glass, plastic, silicon chip, and air. One of the two phases can dissolve the reactants of the amplification reaction, which is called the dispersed phase, and the other phase is immiscible with this phase, called the continuous phase or the separated phase. The method of dispersion can be ultrasonic dispersion or dispersion using a liquid operating system. The liquid operating system can be a microfluidic chip. The method of using a microfluidic chip to generate interconnected dispersed phase micro-droplets is multiphase flow method, electrowetting method, thermal capillary method, dielectrophoresis method, etc. The principle of the multiphase flow method is that through the unique design of the fluid microchannel structure and the control of the fluid velocity, the interaction of the shear force, viscous force and surface tension between the liquid flows is used to make the dispersed phase fluid flow locally in the microchannel. A velocity gradient is generated, which is split to generate micro-droplets, and the generated micro-droplets are evenly distributed in the continuous phase immiscible with the dispersed phase, forming a monodisperse system. In order to reduce surface tension and generate stable micro-droplets, surfactants can also be added to the liquid stream. The channels of the microfluidic chip can be T-shaped, Y-shaped, or cross-shaped. By adjusting the flow rate of the continuous phase and the flow ratio of the continuous phase to the dispersed phase, the formation speed of micro-droplets and the size of the formed micro-droplets can be controlled, thereby controlling the size of the independent reaction system and the number of nucleic acid molecules in each reaction system . The content of the nucleic acid substance in each reaction system is 0 to 50 molecules, preferably 0-20 molecules, more preferably 0-10 molecules. When the system is larger, more products can be produced, and the fewer the number of nucleic acid molecules, the smaller the interaction in a single reaction chamber, and more uniform amplification can be achieved
本发明所述的扩增可以是MDA(多重置换扩增)、PCR(聚合酶链式反应)、RCA(滚环扩增)或MALBAC(多次退火环状循环扩增)。The amplification described in the present invention can be MDA (Multiple Displacement Amplification), PCR (Polymerase Chain Reaction), RCA (Rolling Circle Amplification) or MALBAC (Multiple Annealing Circular Amplification).
扩增时间可以为30分钟至40小时，优选为1小时到20小时。本发明中，每个反应体系中用于扩增的原料是有限的，率先“启动”且扩增速度较快的分子的扩增速度在一段时间后会趋于平稳，因此需要足够长的扩增时间使得反应体系的反应都趋于平稳，才能达到均匀扩增的目的。不同的扩增方法(如MDA、PCR、RCA、MALBAC)和不同的反应体系所需的最小扩增时间不同，扩增时间可通过常规实验手段进行选择。传统的扩增方法中，扩增时间的延长会加剧扩增偏倚，因此实验中通常会控制扩增时间。本发明的方法可有效避免这一问题，允许通过延长扩增时间的手段获得更大量的分子便于下游分析。The amplification time may be 30 minutes to 40 hours, preferably 1 hour to 20 hours. In the present invention, the raw materials used for amplification in each reaction system are limited, and the amplification speed of molecules that are "started" first and have a faster amplification speed will tend to be stable after a period of time, so a sufficiently long amplification time is required. The increase time makes the reaction of the reaction system tend to be stable, so as to achieve the purpose of uniform amplification. Different amplification methods (such as MDA, PCR, RCA, MALBAC) and different reaction systems require different minimum amplification times, and the amplification time can be selected by conventional experimental means. In the traditional amplification method, the extension of the amplification time will aggravate the amplification bias, so the amplification time is usually controlled in the experiment. The method of the present invention can effectively avoid this problem, allowing a larger amount of molecules to be obtained by prolonging the amplification time for downstream analysis.
本发明的核酸物质包含单链DNA；双链DNA；单链RNA；双链RNA；双链RNA／DNA杂交体；部分杂交的DNA以及RNA；经过酶学、化学、生物学方法处理过的DNA或RNA，如：经过免疫沉淀后提取的DNA、亚硫酸盐处理后的DNA、经过甲基转移酶处理后的DNA、RNA经过逆转录处理后的cDNA等。核酸物质可以不来自于自然界，可以为人工合成的核酸物质或自然界核酸物质的某种化学处理产物，可以来自细胞、亚细胞单位、染色体、核酸分子中的一种或多种，可以来自单细胞，单染色体，单个核酸分子，痕量核酸中的一种或多种。微量核酸物质的质量可以小于10ng，优选小于1ng。The nucleic acid substance of the present invention includes single-stranded DNA; double-stranded DNA; single-stranded RNA; double-stranded RNA; double-stranded RNA/DNA hybrid; partially hybridized DNA and RNA; DNA treated by enzymatic, chemical and biological methods Or RNA, such as: DNA extracted after immunoprecipitation, DNA treated with sulfite, DNA treated with methyltransferase, cDNA after reverse transcription of RNA, etc. Nucleic acid substances may not come from nature, but may be artificially synthesized nucleic acid substances or some chemical processing products of natural nucleic acid substances, may come from one or more of cells, subcellular units, chromosomes, and nucleic acid molecules, and may come from single cells , single chromosome, single nucleic acid molecule, one or more of trace nucleic acids. The mass of trace nucleic acid substances may be less than 10 ng, preferably less than 1 ng.
根据后续实验步骤需要，扩增结束后可以进行产物回收。产物回收的方法可以为从每个分隔反应体系中分别将产物取出后混合或者不混合来进行。也可以通过加入乙醇、异丁醇等破乳剂使两相混合的方法进行。According to the needs of subsequent experimental steps, the product can be recovered after the amplification is completed. The method of product recovery can be carried out by taking out the product from each separate reaction system and then mixing or not mixing. It can also be carried out by adding a demulsifier such as ethanol or isobutanol to mix the two phases.
本发明还提供了一种对微量核酸物质进行全基因组或转录组测序的方法，先使用能够减小未知微量核酸物质整体扩增时产生偏倚的扩增方法进行扩增，再进行产物回收、建库及测序。The present invention also provides a method for sequencing the entire genome or transcriptome of trace nucleic acid substances. First, the amplification method that can reduce the bias in the overall amplification of unknown trace nucleic acid substances is used for amplification, and then the product is recovered and constructed. library and sequencing.
使用本发明的扩增方法相对于微量核酸物质在大体系中的扩增方法，扩增偏倚降低1个数量级以上，扩增结果能够准确反应原始核酸物质中的定量信息，可允许更多的扩增循环，从而获得更多的产物。Compared with the amplification method of trace nucleic acid substances in a large system, using the amplification method of the present invention, the amplification bias is reduced by more than one order of magnitude, the amplification result can accurately reflect the quantitative information in the original nucleic acid substance, and more amplification can be allowed. cycle to obtain more products.
附图说明Description of drawings
以下结合附图及具体实施方式对本发明作进一步的详细描述。The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.
图1为实施例1中形成独立反应室的微流控芯片结构。Fig. 1 is the microfluidic chip structure forming an independent reaction chamber in Example 1.
图2为实施例1中所形成的独立反应室的明场图片，反应室分散在连续的矿物油相中。Fig. 2 is a bright field picture of the independent reaction chamber formed in Example 1, the reaction chamber is dispersed in the continuous mineral oil phase.
图3为实施例1和对比例1中得到的单个人类细胞扩增后的染色体片段拷贝数分布图。FIG. 3 is a graph showing the distribution of chromosome segment copy numbers obtained in Example 1 and Comparative Example 1 after amplification of a single human cell.
具体实施方式Detailed ways
实施例1Example 1
1.单细胞的分离：1. Isolation of single cells:
将细胞吹打成单分散，置于PBS缓冲液放在显微镜下进行观察和操作，将毛细管截断成横截面直径20-30μm的毛细针，利用毛细作用吸取单个细胞，并将细胞转移到新PCR管中。The cells are blown into monodisperse, placed in PBS buffer and placed under a microscope for observation and operation, the capillary is cut into a capillary needle with a cross-sectional diameter of 20-30 μm, and a single cell is absorbed by capillary action, and the cell is transferred to a new PCR tube.
2.单细胞的裂解：2. Lysis of single cells:
配制如下裂解液：Prepare the lysate as follows:
其中Proteinase(Qiagen，#19155)需要事先使用50％甘油配制成10mg／mL的储存液。将4μL裂解液混匀后加入到含有单细胞的PCR管中，在PCR仪中进行如下程序：50℃3小时用于裂解反应，70℃30分钟用于失活蛋白酶。获得的基因组DNA)置于-80℃冰箱中待用。Among them, Proteinase (Qiagen, #19155) needs to be prepared into a 10 mg/mL stock solution with 50% glycerol in advance. Mix 4 μL of the lysate and add it to the PCR tube containing single cells, and perform the following program in the PCR instrument: 50°C for 3 hours for cleavage reaction, 70°C for 30 minutes for protease inactivation. The obtained genomic DNA) was placed in a -80°C refrigerator for use.
3.单细胞的扩增反应溶液的配制：3. Preparation of single cell amplification reaction solution:
使用MDA方法进行扩增，配制如下单细胞的扩增反应溶液：Using the MDA method for amplification, prepare the following single-cell amplification reaction solution:
其中BSA为phi29 polymerase中附带。Among them, BSA is attached to phi29 polymerase.
其中N6primer为由6个随机碱基组成的序列，随机碱基随机选自A、G、C、T中任一个。Wherein N6primer is a sequence composed of 6 random bases, and the random bases are randomly selected from any one of A, G, C, and T.
4.反应室的建立4. Setup of the Reaction Chamber
在微流控芯片中利用多相流法产生直径约为50μm的液滴作为分隔的反应室。微流芯片的管道结构如图1所示。管道呈十字交叉形，上下两条管道和左侧管道连接样品入口，从上下两条管道通入矿物油，从左侧管道通入第3步中配制好的单细胞扩增反应溶液，将0.5个标准大气压的压力施加在样品入口处以推动矿物油相和反应溶液相向右侧的管道流动，油相将反应溶液分隔形成分散在油相中体积为50fL若干独立反应室，每个反应室内分子数为0-5个。反应室的体积可通过调节两相的进样速度和芯片的管道横截面调节。反应室形成的过程如图1所示。反应室的建立完成后如图2所示。In the microfluidic chip, the multiphase flow method is used to generate droplets with a diameter of about 50 μm as separated reaction chambers. The pipeline structure of the microfluidic chip is shown in Figure 1. The pipes are in the shape of a cross, the upper and lower pipes and the left pipe are connected to the sample inlet, the upper and lower pipes are fed with mineral oil, and the left pipe is passed into the single cell amplification reaction solution prepared in step 3, and the 0.5 The pressure of 1 standard atmosphere is applied to the sample inlet to push the mineral oil phase and the reaction solution phase to flow to the right pipe. The oil phase separates the reaction solution to form a number of independent reaction chambers with a volume of 50 fL dispersed in the oil phase. The number of molecules in each reaction chamber 0-5. The volume of the reaction chamber can be adjusted by adjusting the injection speed of the two phases and the channel cross-section of the chip. The process of reaction chamber formation is shown in Figure 1. After the establishment of the reaction chamber is completed, it is shown in Figure 2.
5.扩增反应5. Amplification reaction
使用MDA方法进行扩增。将在油相中分隔好的反应室用移液器置于新的PCR管中，在PCR仪上进行如下程序：30℃6小时用于进行扩增反应，65℃20分钟用于失活使用的Phi29聚合酶。Amplification was performed using the MDA method. Place the reaction chamber separated in the oil phase into a new PCR tube with a pipette, and perform the following program on the PCR machine: 30°C for 6 hours for amplification reaction, 65°C for 20 minutes for inactivation Phi29 polymerase.
6.扩增产物的纯化6. Purification of Amplified Products
扩增反应结束后加入700μL异丁醇，使由油分隔而成的水相反应室相互融合，并通过DNA Clean＆Concentrator kit(Zymo，#D4033)进行纯化，步骤包括：After the amplification reaction, 700 μL of isobutanol was added to fuse the water-phase reaction chambers separated by oil, and the DNA Clean & Concentrator kit (Zymo, #D4033) was used for purification. The steps included:
a)加入70μL的Binding Buffer，15000rpm旋转3分钟，去掉最上层的异丁醇。a) Add 70 μL of Binding Buffer, rotate at 15,000 rpm for 3 minutes, and remove the top layer of isobutanol.
b)将下层的溶液均转移到硅胶膜的柱子，15000prm旋转一分钟，去掉液相。b) Transfer the solution in the lower layer to a column with a silica gel membrane, rotate at 15000 rpm for one minute, and remove the liquid phase.
c)加入350μl Wash Buffer，15000rpm旋转一分钟，去掉液相。c) Add 350μl Wash Buffer, rotate at 15000rpm for one minute, and remove the liquid phase.
d)重复步骤c两次。d) Repeat step c twice.
e)将硅胶柱转移到新的1.5mL离心管中(Eppendorf#0030120.086)，加入20μL的TE buffer，静置5分钟后，15000rpm旋转一分钟，附着在硅胶膜上的DNA将会被洗脱，置于-80℃冰箱中待用。e) Transfer the silica gel column to a new 1.5mL centrifuge tube (Eppendorf#0030120.086), add 20μL of TE buffer, let it stand for 5 minutes, then rotate at 15000rpm for 1 minute, the DNA attached to the silica gel membrane will be eluted, Store in -80°C refrigerator for later use.
7.DNA的破碎7. Fragmentation of DNA
由于高通量测序的读长限制，需要破碎生成的DNA至较短的片段。Due to the read length limitation of high-throughput sequencing, the generated DNA needs to be fragmented into shorter fragments.
取出步骤6获得的产物100ng，用130μl的Low TE缓冲液进行稀释，用Covaris超声仪进行破碎，使用程序为自带的DNA-200。用DNA Clean＆Concentrator kit(Zymo，#D4033)纯化，主要步骤包括：Take out 100 ng of the product obtained in step 6, dilute it with 130 μl of Low TE buffer, and break it with a Covaris ultrasonic instrument, using the included DNA-200 program. Purify with DNA Clean & Concentrator kit (Zymo, #D4033), the main steps include:
a)加入260μL的Binding Buffer，混合均匀后的溶液均转移到硅胶膜的柱子，15000prm旋转一分钟，去掉液相。a) Add 260 μL of Binding Buffer, transfer the mixed solution to a column with silica gel membrane, rotate at 15000 rpm for one minute, and remove the liquid phase.
b)加入350μl Wash Buffer，15000rpm旋转一分钟，去掉液相。b) Add 350μl Wash Buffer, rotate at 15000rpm for one minute, and remove the liquid phase.
c)重复步骤b两次。c) Repeat step b twice.
d)将硅胶柱转移到新的1.5mL离心管中(Eppendorf#0030120.086)，加入20μL的蒸馏水，静置5分钟后，15000rpm旋转一分钟，附着在硅胶膜上的DNA将会被洗脱，保存于-20℃冰箱待用。d) Transfer the silica gel column to a new 1.5mL centrifuge tube (Eppendorf#0030120.086), add 20μL of distilled water, let it stand for 5 minutes, then rotate it at 15000rpm for 1 minute, the DNA attached to the silica gel membrane will be eluted, save Store in -20°C refrigerator for later use.
8.测序文库的建立：8. Establishment of sequencing library:
使用UltraTM DNA Library Prep Kit for Illumina(NEB，#N7370)进行测序文库的建立。主要步骤包括：use UltraTM DNA Library Prep Kit for Illumina (NEB, #N7370) was used to establish the sequencing library. The main steps include:
a)DNA的补齐。由于DNA破碎时候5’和3’端有一些不相互配对的区域，在这一步进行补齐或者切除，使得所有DNA均变成正常双链DNA分子。a) Completion of DNA. Since there are some unpaired regions at the 5' and 3' ends of the DNA when it is broken, it is filled or excised at this step, so that all DNA becomes a normal double-stranded DNA molecule.
配制如下反应体系：Prepare the following reaction system:
混合均匀后在PCR仪上执行如下程序：After mixing evenly, perform the following procedures on the PCR instrument:
20℃30分钟用于反应，65℃30分钟用于失活末端修复的酶30 minutes at 20°C for reaction, 30 minutes at 65°C for inactivation of end-repair enzymes
b)连接测序接头b) Ligation of sequencing adapters
将接头按照1：10进行稀释并制如下反应体系：Dilute the joint according to 1:10 and make the following reaction system:
混合均匀后在PCR仪上执行如下程序：After mixing evenly, perform the following procedures on the PCR instrument:
20℃30分钟用于反应。20°C for 30 minutes for the reaction.
c)之后的片段选择和扩增步骤同NEB公司给该产品提供的实验方案相同(实验方案版本号为Version1.213年2月版本)，建立好的DNA文库即可以进行测序。c) The subsequent fragment selection and amplification steps are the same as the experimental protocol provided by NEB Company for this product (the experimental protocol version number is Version 1.213 in February), and the established DNA library can be sequenced.
9.测序及生物信息学分析9. Sequencing and bioinformatics analysis
我们使用Illumine公司的miSeq测序仪进行测序，产生的读数是50个碱基长度的片段。对于不小于1,000,000,000碱基数目的CNV检测，每个样品需要0.01倍于其物种基因组大小的测序深度，在此每个样品获得了大约600,000条读数，大约为0.03G的测序深度。得到了原始的测序图片数据之后，将其转换成可以阅读的碱基序列，然后删除掉序质量过低的数据之后将序列通过比对软件比对到参考基因组上，将基因组分成大小相同的1,000,000,000碱基长度的片段，计算每个片段总共被测得的次数，除以一个均一化值：(片段大小*测序得到的总碱基数)／基因组总碱基数据，即为每个片段的均一化的拷贝数目。通过单细胞扩增再测序得到的对于人类基因组不同的片段和相应得到的拷贝数据作图，如图3上部分所示。其中一些为0值的片段为参考基因组未收录的部分，一般位于中心体附近或者染色体的两端，此处重复序列较多，无法知道真实的序列，因此将其排除。图中每个点对应的纵坐标的值为均一化后的拷贝数，横坐标对应的是相应的染色体编号的1,000,000,000个碱基的片段。实施例1的不同片段的拷贝数之间的差异较小，常染色体都平均为2个拷贝，X染色体和Y染色体均为一个拷贝，这和已知的知识是一致的。We performed sequencing using the Illumina miSeq sequencer, which generated reads as fragments of 50 bases in length. For CNV detection with a base number of not less than 1,000,000,000 bases, each sample requires a sequencing depth of 0.01 times the genome size of its species, where about 600,000 reads were obtained for each sample, with a sequencing depth of approximately 0.03G. After obtaining the original sequencing image data, convert it into a base sequence that can be read, then delete the out-of-sequence data with low quality, and then compare the sequence to the reference genome through the comparison software, and divide the genome into 1,000,000,000 of the same size For fragments of base length, calculate the total number of times each fragment is measured, and divide it by a normalization value: (fragment size * total number of bases obtained by sequencing) / total genome data, that is, the uniformity of each fragment number of copies. The different fragments of the human genome obtained by single-cell amplification and re-sequencing and the corresponding copy data are plotted, as shown in the upper part of FIG. 3 . Some fragments with a value of 0 are not included in the reference genome, and are generally located near the centrosome or at both ends of the chromosome. There are many repeated sequences here, and the real sequence cannot be known, so they are excluded. The value of the ordinate corresponding to each point in the figure is the normalized copy number, and the abscissa corresponds to the fragment of 1,000,000,000 bases of the corresponding chromosome number. The difference between the copy numbers of different fragments in Example 1 is small, the autosomes have an average of 2 copies, and both the X chromosome and the Y chromosome have one copy, which is consistent with the known knowledge.
实施例2Example 2
建立反应室时，将10μL配制好的单细胞扩增反应溶液与100μL矿物油混合，于500HZ频率超声震荡3分钟，形成分散在油相中的水相反应室，反应室体系为10fL，反应室的体积可以通过控制超声的时间和频率进行改变。除反应室的建立步骤，其他步骤均与实施例1相同。When establishing the reaction chamber, mix 10 μL of the prepared single-cell amplification reaction solution with 100 μL of mineral oil, and ultrasonically oscillate at a frequency of 500 Hz for 3 minutes to form a water-phase reaction chamber dispersed in the oil phase. The reaction chamber system is 10 fL, and the reaction chamber The volume can be changed by controlling the time and frequency of ultrasound. Except for the steps of setting up the reaction chamber, other steps are the same as in Example 1.
对比例1Comparative example 1
步骤3配制好的反应溶液直接在10μL体系中进行扩增反应，扩增反应结束进行DNA的纯化，纯化步骤为Zymo公司给定的实验方案版本号1.0一致，省去了建立反应室的步骤。单细胞的分离、裂解、反应溶液的配制、扩增反应、DNA的破碎、测序文库的建立、比对的步骤均与实施例1相同。The reaction solution prepared in step 3 was directly amplified in the 10 μL system, and the DNA was purified after the amplification reaction. The purification steps were consistent with the version number 1.0 of the experimental protocol given by Zymo, and the step of establishing a reaction chamber was omitted. The steps of single cell separation, lysis, preparation of reaction solution, amplification reaction, fragmentation of DNA, establishment of sequencing library, and comparison are all the same as in Example 1.
根据比对结果对于人类基因组不同的片段和相应得到的均一化的拷贝数据作图，如图3下部分所示。从对比例1的图来看，不同片段的拷贝数之间的差异很大，常染色体的拷贝数在1-4之间都有分布，完全不可能从该图中直观的看出每条染色体的拷贝数，说明扩增后的结果已经无法代表扩增前单细胞的状态，存在较多的扩增偏倚。实施例1的染色体片段拷贝数的变异系数(常染色体片段均一化拷贝数的标准差／常染色体片段均一化拷贝数的平均值)为0.04，与对比例1的变异系数0.4相比，扩增偏倚降低10倍。According to the comparison results, the different fragments of the human genome and the corresponding normalized copy data were plotted, as shown in the lower part of FIG. 3 . From the figure of Comparative Example 1, the copy numbers of different fragments vary greatly, and the copy numbers of autosomes are distributed between 1-4. It is completely impossible to see each chromosome intuitively from this figure copy number, indicating that the results after amplification can no longer represent the state of single cells before amplification, and there are more amplification biases. The coefficient of variation (the standard deviation of the normalized copy number of the autosomal segment/the average value of the normalized copy number of the autosomal segment) of the chromosome segment copy number of Example 1 is 0.04, compared with the coefficient of variation 0.4 of Comparative Example 1, the amplification 10-fold reduction in bias.
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| CN201310562192.0ACN104630202A (en) | 2013-11-13 | 2013-11-13 | Amplification method capable of decreasing bias generation during trace nucleic acid substance entire amplification |
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|---|---|---|---|
| CN201310562192.0ACN104630202A (en) | 2013-11-13 | 2013-11-13 | Amplification method capable of decreasing bias generation during trace nucleic acid substance entire amplification |
| Publication Number | Publication Date |
|---|---|
| CN104630202Atrue CN104630202A (en) | 2015-05-20 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310562192.0APendingCN104630202A (en) | 2013-11-13 | 2013-11-13 | Amplification method capable of decreasing bias generation during trace nucleic acid substance entire amplification |
| Country | Link |
|---|---|
| CN (1) | CN104630202A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925675A (en)* | 2016-04-26 | 2016-09-07 | 序康医疗科技（苏州）有限公司 | Method for amplifying dna |
| CN106929595A (en)* | 2017-04-28 | 2017-07-07 | 上海亿康医学检验所有限公司 | A kind of system and method for identifying embryo's balanced translocation carrier state |
| CN107058291A (en)* | 2017-05-03 | 2017-08-18 | 东南大学 | Whole genome amplification method based on elongated reaction chamber |
| CN109439734A (en)* | 2018-10-17 | 2019-03-08 | 东南大学 | A kind of whole genome amplification method based on Ago-Gel medium |
| CN112251500A (en)* | 2020-10-21 | 2021-01-22 | 东南大学 | A method that can improve the efficiency of micro-nucleic acid amplification |
| US12181650B2 (en) | 2019-12-10 | 2024-12-31 | Enumerix, Inc. | Methods and systems for three-dimensional lightsheet imaging |
| US12252745B2 (en) | 2021-09-02 | 2025-03-18 | Enumerix, Inc. | Detection and digital quantitation of multiple targets |
| US12265088B2 (en) | 2021-03-05 | 2025-04-01 | Countable Labs, Inc. | Systems and methods for generating droplets and performing digital analyses |
| US12319964B2 (en) | 2021-12-20 | 2025-06-03 | Countable Labs, Inc. | Detection and digital quantitation of multiple targets |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101171345A (en)* | 2005-04-12 | 2008-04-30 | 454生命科学公司 | Methods for determining sequence variants using ultra-deep sequencing |
| CN101278058A (en)* | 2005-06-23 | 2008-10-01 | 科因股份有限公司 | Improved strategies for sequencing complex genomes using high throughput sequencing technologies |
| CN101365803A (en)* | 2005-12-22 | 2009-02-11 | 关键基因股份有限公司 | Improved strategies for transcript profiling using high throughput sequencing technologies |
| CN101374963A (en)* | 2005-12-22 | 2009-02-25 | 凯津公司 | Method for AFLP-based high throughput polymorphism detection |
| CN101432438A (en)* | 2006-04-04 | 2009-05-13 | 凯津公司 | High throughput detection of molecular markers based on AFLP and high throughput sequencing |
| CN101506378A (en)* | 2006-06-19 | 2009-08-12 | 约翰·霍普金斯大学 | Single-molecule PCR on microparticles in water-in-oil emulsions |
| CN101736087A (en)* | 2010-01-28 | 2010-06-16 | 华东医学生物技术研究所 | Method for digitally detecting micro-mutation by using micro-emulsion clone amplified bound water gel microsphere chip |
| CN102212614A (en)* | 2003-01-29 | 2011-10-12 | 454生命科学公司 | Methods of amplifying and sequencing nucleic acids |
| CN102311948A (en)* | 2010-07-07 | 2012-01-11 | 霍夫曼-拉罗奇有限公司 | Clone in the emulsion increases in advance |
| CN102625850A (en)* | 2009-04-03 | 2012-08-01 | 蒂莫西·Z·刘 | Multiplex nucleic acid detection method and system |
| CN102943111A (en)* | 2012-11-16 | 2013-02-27 | 北京爱普益生物科技有限公司 | Application of high-pass DNA (Deoxyribonucleic Acid) sequencing method on determination of short tandem repeat gene locus in human genome and method |
| CN103119165A (en)* | 2010-09-27 | 2013-05-22 | 威泊根私人有限公司 | A method for making an enriched library |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212614A (en)* | 2003-01-29 | 2011-10-12 | 454生命科学公司 | Methods of amplifying and sequencing nucleic acids |
| CN101171345A (en)* | 2005-04-12 | 2008-04-30 | 454生命科学公司 | Methods for determining sequence variants using ultra-deep sequencing |
| CN101278058A (en)* | 2005-06-23 | 2008-10-01 | 科因股份有限公司 | Improved strategies for sequencing complex genomes using high throughput sequencing technologies |
| CN101365803A (en)* | 2005-12-22 | 2009-02-11 | 关键基因股份有限公司 | Improved strategies for transcript profiling using high throughput sequencing technologies |
| CN101374963A (en)* | 2005-12-22 | 2009-02-25 | 凯津公司 | Method for AFLP-based high throughput polymorphism detection |
| CN101432438A (en)* | 2006-04-04 | 2009-05-13 | 凯津公司 | High throughput detection of molecular markers based on AFLP and high throughput sequencing |
| CN101506378A (en)* | 2006-06-19 | 2009-08-12 | 约翰·霍普金斯大学 | Single-molecule PCR on microparticles in water-in-oil emulsions |
| CN102625850A (en)* | 2009-04-03 | 2012-08-01 | 蒂莫西·Z·刘 | Multiplex nucleic acid detection method and system |
| CN101736087A (en)* | 2010-01-28 | 2010-06-16 | 华东医学生物技术研究所 | Method for digitally detecting micro-mutation by using micro-emulsion clone amplified bound water gel microsphere chip |
| CN102311948A (en)* | 2010-07-07 | 2012-01-11 | 霍夫曼-拉罗奇有限公司 | Clone in the emulsion increases in advance |
| CN103119165A (en)* | 2010-09-27 | 2013-05-22 | 威泊根私人有限公司 | A method for making an enriched library |
| CN102943111A (en)* | 2012-11-16 | 2013-02-27 | 北京爱普益生物科技有限公司 | Application of high-pass DNA (Deoxyribonucleic Acid) sequencing method on determination of short tandem repeat gene locus in human genome and method |
| Title |
|---|
| MICHIHIKO NAKANO 等: "Single-Molecule Reverse Transcription Polymerase Chain Reaction Using Water-in-Oil Emulsion", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》* |
| TAKAAKI KOJIMA 等: "PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets", 《NUCLEIC ACIDS RESEARCH》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017186117A1 (en)* | 2016-04-26 | 2017-11-02 | 序康医疗科技（苏州）有限公司 | Dna amplification method |
| CN105925675B (en)* | 2016-04-26 | 2020-06-05 | 序康医疗科技（苏州）有限公司 | Methods of Amplifying DNA |
| CN105925675A (en)* | 2016-04-26 | 2016-09-07 | 序康医疗科技（苏州）有限公司 | Method for amplifying dna |
| CN106929595A (en)* | 2017-04-28 | 2017-07-07 | 上海亿康医学检验所有限公司 | A kind of system and method for identifying embryo's balanced translocation carrier state |
| CN107058291A (en)* | 2017-05-03 | 2017-08-18 | 东南大学 | Whole genome amplification method based on elongated reaction chamber |
| CN109439734A (en)* | 2018-10-17 | 2019-03-08 | 东南大学 | A kind of whole genome amplification method based on Ago-Gel medium |
| CN109439734B (en)* | 2018-10-17 | 2022-01-28 | 东南大学 | Whole genome amplification method based on agarose gel medium |
| US12181650B2 (en) | 2019-12-10 | 2024-12-31 | Enumerix, Inc. | Methods and systems for three-dimensional lightsheet imaging |
| CN112251500A (en)* | 2020-10-21 | 2021-01-22 | 东南大学 | A method that can improve the efficiency of micro-nucleic acid amplification |
| US12265088B2 (en) | 2021-03-05 | 2025-04-01 | Countable Labs, Inc. | Systems and methods for generating droplets and performing digital analyses |
| US12270815B2 (en) | 2021-03-05 | 2025-04-08 | Countable Labs, Inc. | Systems and methods for generating droplets and performing digital analyses |
| US12252745B2 (en) | 2021-09-02 | 2025-03-18 | Enumerix, Inc. | Detection and digital quantitation of multiple targets |
| US12319964B2 (en) | 2021-12-20 | 2025-06-03 | Countable Labs, Inc. | Detection and digital quantitation of multiple targets |
| Publication | Publication Date | Title |
|---|---|---|
| CN104630202A (en) | Amplification method capable of decreasing bias generation during trace nucleic acid substance entire amplification | |
| US11097247B2 (en) | Multiple beads per droplet resolution | |
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