CN109423510B - Method for detecting RCA product and application thereof - Google Patents
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Abstract
Description
Translated fromChinese技术领域technical field
本发明涉及分子生物学技术领域，涉及一种检测RCA产物的方法及应用，具体地，涉及一种检测滚环扩增产物的方法及筛选DNA聚合酶的方法。The invention relates to the technical field of molecular biology, to a method and application for detecting RCA products, and in particular, to a method for detecting rolling circle amplification products and a method for screening DNA polymerases.
背景技术Background technique
滚环扩增(RCA)或多重链置换扩增(MDA)具有快速、准确、所需模板量少等特点，目前已成为生物医学技术和生物纳米技术领域重要的研究手段。Phi29是一种具有链置换能力的DNA聚合酶，已经广泛应用于RCA和MDA。然而，RCA或MDA并不像PCR反应那样，可以通过控制循环数来控制扩增产物的拷贝数。这主要是因为：(1)模板本身具有不同特征，如不同的碱基含量，poly结构等，将导致phi29在扩增中对于不同的模板产生不一样的扩增效率；(2)phi29酶是一种稳定性较低的酶，随反应时间的延长，活性会逐渐下降，因而难以较准确地评估，一定时间内RCA/MDA产物的拷贝数目。基于上述两点，RCA和MDA的产物往往均一性不够，严重影响了这两种技术的应用。Rolling circle amplification (RCA) or multiple strand displacement amplification (MDA) has the characteristics of rapidity, accuracy, and small amount of template required, and has now become an important research method in the fields of biomedical technology and bio-nanotechnology. Phi29 is a DNA polymerase with strand displacement ability that has been widely used in RCA and MDA. However, RCA or MDA are not like PCR reactions, which can control the copy number of the amplified product by controlling the number of cycles. This is mainly because: (1) the template itself has different characteristics, such as different base content, poly structure, etc., which will cause phi29 to produce different amplification efficiencies for different templates during amplification; (2) phi29 enzyme is a For an enzyme with low stability, the activity will gradually decrease with the prolongation of reaction time, so it is difficult to accurately assess the number of copies of RCA/MDA products in a certain period of time. Based on the above two points, the products of RCA and MDA are often not uniform enough, which seriously affects the application of these two technologies.
现有的检测RCA或MDA产物方法主要通过电泳，如琼脂糖电泳和脉冲电泳。但RCA产物普遍分子量较大，应用琼脂糖电泳时，大分子产物容易堆积在点样孔处，导致电泳失败。应用脉冲电泳虽然解决了产物滞留点样孔的缺点，但其存在操作复杂、耗时长的劣势。此外，电泳技术对RCA产物大小的定量较为粗糙，对于精确研究RCA产物均一性的意义不大。不仅如此，也有通过荧光定量PCR的技术来检测RCA产物的拷贝数，这种方法虽然可以做到定量，但是此方法是基于确定模板或是将RCA产物当成一个整体来检测的，对于复杂模板的扩增没有指导意义。Existing methods for detecting RCA or MDA products are mainly by electrophoresis, such as agarose electrophoresis and pulse electrophoresis. However, RCA products generally have large molecular weights, and when agarose electrophoresis is used, macromolecular products are easy to accumulate in the spotting wells, resulting in failure of electrophoresis. Although the application of pulse electrophoresis solves the shortcomings of product retention in the spot holes, it has the disadvantages of complicated operation and long time. In addition, the quantification of the size of the RCA product by electrophoresis is relatively rough, and it is of little significance for the precise study of the homogeneity of the RCA product. Not only that, there is also a technique to detect the copy number of RCA products by fluorescence quantitative PCR. Although this method can be quantitative, this method is based on determining the template or detecting the RCA products as a whole. Amplification is not instructive.
RCA产物是一种多拷贝的单链DNA序列，因内部DNA序列的碱基间的相互作用力，可以形成类似“球形“结构。片段化的基因组DNA加上接头序列，并环化形成单链环状DNA，随后使用滚环扩增技术可将单链环状DNA扩增多个数量级，所产生的扩增产物称为DNA纳米球(DNA nano ball,DNB)。由此可知，DNB实质上是一种典型的RCA产物，具有RCA产物的特征。因模板DNA碱基序列的差异，phi29聚合酶活性差异及反应条件的不同，RCA产物通常会表现出不同的大小甚至形态特征。因RCA产物是分子量巨大的单链DNA序列，传统的检测方法，如凝胶电泳法，及qubit定量分析仪，难以分辨不同RCA产物的大小，及一个反应体系中产物的大小分布。纳米粒径分析仪采用的是动态散射技术，根据不同粒径纳米颗粒的布朗运动状态的不同，依托现有的纳米颗粒粒径检测设备和配套的荧光信号处理软件，定量纳米颗粒的大小。而对于并不是纳米颗粒特征的RCA产物是否可以采用粒径分析仪进行检测和区分，目前为止仍未有相关的报道。The RCA product is a multi-copy single-stranded DNA sequence that can form a "spherical"-like structure due to the interaction between the bases of the internal DNA sequence. Fragmented genomic DNA is added with an adaptor sequence, and circularized to form single-stranded circular DNA. The single-stranded circular DNA can then be amplified by multiple orders of magnitude using rolling circle amplification technology. The resulting amplification product is called DNA nanometer. Ball (DNA nano ball, DNB). It can be seen that DNB is essentially a typical RCA product with the characteristics of RCA products. Due to the differences in the base sequence of the template DNA, the activity of the phi29 polymerase and the reaction conditions, the RCA products usually show different sizes and even morphological characteristics. Because RCA products are single-stranded DNA sequences with huge molecular weight, traditional detection methods, such as gel electrophoresis and qubit quantitative analyzer, are difficult to distinguish the size of different RCA products and the size distribution of products in a reaction system. The nanoparticle size analyzer adopts dynamic scattering technology. According to the different Brownian motion states of nanoparticles with different sizes, relying on the existing nanoparticle size detection equipment and supporting fluorescence signal processing software, the size of nanoparticles can be quantified. As for whether RCA products that are not characteristic of nanoparticles can be detected and differentiated by a particle size analyzer, there is no relevant report so far.
因此，有必要开发一种快速准确地检测RCA产物的方法，方便有效地优化RCA反应体系。Therefore, it is necessary to develop a method for rapid and accurate detection of RCA products, and to optimize the RCA reaction system conveniently and efficiently.
发明内容SUMMARY OF THE INVENTION
针对目前检测RCA产物的手段有限，检测方法操作复杂、准确性低、耗时长的问题，本发明提供了一种检测RCA产物的方法和应用，所述方法配合荧光信号处理软件，可以直接分析RCA产物的粒径，评价RCA产物的均一性，具有快速、准确且操作简便等优点。Aiming at the problems of limited means for detecting RCA products at present, complicated operation, low accuracy and long time-consuming of the detection method, the present invention provides a method and application for detecting RCA products. The method cooperates with fluorescence signal processing software, and can directly analyze RCA The particle size of the product is used to evaluate the uniformity of the RCA product, which has the advantages of rapidity, accuracy and simple operation.
为达此目的，本发明采用了以下技术方案：For this purpose, the present invention has adopted the following technical solutions:
第一方面，本发明提供了一种检测RCA产物的方法，包括如下步骤：In a first aspect, the invention provides a method for detecting an RCA product, comprising the steps of:
(1)以环状DNA分子为模板，经过滚环扩增，获得RCA产物；(1) Using the circular DNA molecule as a template, through rolling circle amplification, the RCA product is obtained;
(2)采用粒径分析仪对所述RCA产物进行检测，获得粒径分析结果。(2) using a particle size analyzer to detect the RCA product to obtain particle size analysis results.
本发明中，创新性的将粒径分析仪用于RCA产物的检测，发明人意外发现，通过粒径分析仪的检测可以区分不同分子量大小的RCA产物，通过得到的粒径分布图能够准确区分不同分子量大小的RCA产物，从而提高RCA产物的均一性，优化RCA产物的制备体系。In the present invention, the particle size analyzer is innovatively used for the detection of RCA products. The inventor unexpectedly found that RCA products with different molecular weights can be distinguished by the detection of the particle size analyzer, and the obtained particle size distribution diagram can accurately distinguish RCA products with different molecular weights, thereby improving the homogeneity of RCA products and optimizing the preparation system of RCA products.
根据本发明，步骤(1)所述环状DNA分子滚环扩增的时间不同会产生不同分子量大小的RCA产物，发明人发现，通过本发明方法可以同时检测不同分子量大小的RCA产物，本发明环状DNA分子滚环扩增的时间为5-60min，例如可以是5min、10min、20min、30min、40min、50min或60min。According to the present invention, the time of rolling circle amplification of the circular DNA molecule in step (1) is different, and RCA products with different molecular weights will be generated. The time for the rolling circle amplification of the circular DNA molecule is 5-60 minutes, for example, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes.
需要说明的是，环状DNA分子滚环扩增的时间主要取决于反应所采用的DNA聚合酶。由于聚合酶不同，反应的速度不同，滚环扩增所需的反应时间也不尽相同。即，野生型DNA聚合酶与突变型DNA聚合酶，或者不同突变型DNA聚合酶之间，在进行滚环扩增时，所需要的反应时间可能是不相同的，本领域技术人员可以根据需要调整滚环扩增反应的时间。It should be noted that the time of rolling circle amplification of circular DNA molecules mainly depends on the DNA polymerase used in the reaction. Due to different polymerases, the reaction speed is different, and the reaction time required for rolling circle amplification is also different. That is, the reaction time required for rolling circle amplification may be different between wild-type DNA polymerase and mutant DNA polymerase, or between different mutant DNA polymerases, and those skilled in the art can perform rolling circle amplification as required. Adjust the time of the rolling circle amplification reaction.
根据本发明，在步骤(1)所述滚环扩增之后，还包括加入荧光分子修饰的探针与所述RCA产物杂交的步骤。According to the present invention, after the rolling circle amplification in step (1), the step of adding a probe modified by a fluorescent molecule to hybridize with the RCA product is further included.
优选地，所述荧光分子为ROX、FAM、HEX、VIC、Cy5或Cy3中的任意一种或至少两种的组合，优选为ROX。Preferably, the fluorescent molecule is any one or a combination of at least two of ROX, FAM, HEX, VIC, Cy5 or Cy3, preferably ROX.
本发明中，所述检测RCA产物的荧光分子修饰的探针的核苷酸序列如SEQID NO.1所示，所述SEQ ID NO.1所示的核苷酸序列如下：5’-ROXGCTCACAGAACGACATGGCTACGATCCGACTT-3’.In the present invention, the nucleotide sequence of the fluorescent molecule-modified probe for detecting RCA products is shown in SEQ ID NO.1, and the nucleotide sequence shown in SEQ ID NO.1 is as follows: 5'-ROXGCTCACAGAACGACATGGCTACGATCCGACTT- 3'.
根据本发明，所述荧光分子修饰的探针的加入量为0.5-5μM，例如可以是0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1μM、1.1μM、1.2μM、1.3μM、1.5μM、1.8μM、2μM、2.3μM、2.5μM、2.8μM、3μM、3.2μM、3.5μM、3.8μM、4μM、4.2μM、4.5μM、4.8μM或5μM，优选为1μM。According to the present invention, the added amount of the fluorescent molecule-modified probe is 0.5-5 μM, for example, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.5 μM μM, 1.8 μM, 2 μM, 2.3 μM, 2.5 μM, 2.8 μM, 3 μM, 3.2 μM, 3.5 μM, 3.8 μM, 4 μM, 4.2 μM, 4.5 μM, 4.8 μM or 5 μM, preferably 1 μM.
根据本发明，所述RCA产物为DNA纳米球(DNB)，所述DNB为DNA纳米球(DNAnanoball,DNB)，是使用RCA技术将单链环状DNA扩增多个数量级，所产生的扩增产物。本发明采用控制不同的反应时间来制备不同分子量大小的DNB。According to the present invention, the RCA product is DNA nanoball (DNB), and the DNB is DNA nanoball (DNB), which is the amplification of single-stranded circular DNA by multiple orders of magnitude using RCA technology, and the resulting amplification product. The present invention prepares DNBs with different molecular weights by controlling different reaction times.
根据本发明，所述粒径分析结果包括RCA产物的分子大小、RCA产物的分子分布或RCA产物的均一性中的任意一种或至少两种的组合。According to the present invention, the particle size analysis result includes any one or a combination of at least two of the molecular size of the RCA product, the molecular distribution of the RCA product, or the homogeneity of the RCA product.
在一个具体的实施例中，步骤(1)具体包括：取环化后的DNA文库20μL到PCR管内，加入20μL DNB制备缓冲液，缓冲液中所用的扩增引物序列与环化DNA文库上的序列互补配对。漩涡振荡器震荡混匀，迷你离心机离心后置于PCR仪中反应，反应条件为：95℃1min，65℃1min，40 1min，4℃保持；当温度达到4℃时取出PCR管，迷你离心机离心后加入40μL DNB聚合酶混合液和4μL DNB聚合酶混合液II，漩涡振荡器震荡混匀，迷你离心机离心后即刻置于PCR仪中开始反应，反应条件：30℃20min，4℃保持；反应结束后，加入20μL终止缓冲液，即获得RCA产物。In a specific embodiment, step (1) specifically includes: taking 20 μL of the circularized DNA library into a PCR tube, adding 20 μL of DNB preparation buffer, and the amplification primer sequences used in the buffer are the same as those on the circularized DNA library. Sequence complementary pairing. Vortex shaker to mix evenly, centrifuge in a mini centrifuge and place it in a PCR machine for reaction. The reaction conditions are: 95°C for 1 min, 65°C for 1 min, 40 for 1 min, and keep at 4°C; when the temperature reaches 4°C, take out the PCR tube and mini centrifuge After centrifugation, 40 μL of DNB polymerase mixture and 4 μL of DNB polymerase mixture II were added, and the vortex shaker was shaken to mix evenly. Immediately after centrifugation in the mini-centrifuge, placed in the PCR machine to start the reaction. Reaction conditions: 30 °C for 20 min, kept at 4 °C ; After the reaction, add 20 μL of stop buffer to obtain the RCA product.
根据本发明，在步骤(1)之前，还包括制备环状DNA分子的步骤，所述环状DNA分子的制备方法为本领域的常规方法，本领域技术人员可以根据需要进行选择，在此不做特殊限定。According to the present invention, before step (1), a step of preparing a circular DNA molecule is also included, and the preparation method of the circular DNA molecule is a conventional method in the field, and those skilled in the art can choose according to the needs. Make special restrictions.
根据被本发明，在步骤(2)之前，还包括将步骤(1)获得的RCA产物进行稀释的步骤。According to the present invention, before step (2), the step of diluting the RCA product obtained in step (1) is further included.
根据被本发明，采用稀释液进行稀释，所述稀释液为PBS和/或加载缓冲液，优选为PBS和加载缓冲液的组合。According to the present invention, the dilution is performed using a diluent, the diluent being PBS and/or loading buffer, preferably a combination of PBS and loading buffer.
根据被本发明，所述加载缓冲液为非离子表面活性剂，优选为聚氧乙烯聚氧丙烯嵌段共聚物，进一步优选为泊洛沙姆188。According to the present invention, the loading buffer is a nonionic surfactant, preferably a polyoxyethylene polyoxypropylene block copolymer, more preferably poloxamer 188.
根据本发明，所述稀释具体包括：将RCA产物与PBS缓冲液混合得到混合液，再向所述混合液中加入所述加载缓冲液。According to the present invention, the dilution specifically includes: mixing the RCA product with a PBS buffer to obtain a mixed solution, and then adding the loading buffer to the mixed solution.
根据本发明，所述RCA产物与所述PBS缓冲液的体积比为1:(9-9999)，例如可以是1:9、1:10、1:12、1:15、1:20、1:30、1:40、1:50、1:60、1:70、1:80、1:90、1:99、1:100、1:120、1:150、1:180、1:200、1:250、1:300、1:350、1:400、450、1:480、1:499、1:500、1:600、1:700、1:800、1:900、1:950、1:999、1:1000、1:1100、1:1200、1:1500、1:1800、1:2000、1:2500、1:3000、1:3500、1:4000、1:4500、1:5000、1:5500、1:6000、1:6500、1:7000、1:7500、1:8000、1:8500、1:9000、1:9500或1:9999，优选为1:(99-999)，进一步优选为1:99。According to the present invention, the volume ratio of the RCA product to the PBS buffer is 1:(9-9999), such as 1:9, 1:10, 1:12, 1:15, 1:20, 1 :30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:99, 1:100, 1:120, 1:150, 1:180, 1:200 , 1:250, 1:300, 1:350, 1:400, 450, 1:480, 1:499, 1:500, 1:600, 1:700, 1:800, 1:900, 1:950 , 1:999, 1:1000, 1:1100, 1:1200, 1:1500, 1:1800, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1 :5000, 1:5500, 1:6000, 1:6500, 1:7000, 1:7500, 1:8000, 1:8500, 1:9000, 1:9500 or 1:9999, preferably 1:(99- 999), more preferably 1:99.
根据本发明，所述混合液与所述加载缓冲液的体积比为(100-10000):1，例如可以是100:1、110:1、120:1、150:1、180:1、200:1、230:1、250:1、280:1、300:1、350:1、400:1、450:1、500:1、550:1、600:1、650:1、700:1、750:1、800:1、850:1、900:1、950:1、1000:1、1100:1、1200:1、1500:1、2000:1、2500:1、3000:1、3500:1、4000:1、4500:1、5000:1、5500:1、6000:1、6500:1、7000:1、7500:1、8000:1、8500:1、9000:1、9500:1或10000:1，优选为(100-1000):1，进一步优选为100:1。According to the present invention, the volume ratio of the mixed solution to the loading buffer is (100-10000):1, for example, 100:1, 110:1, 120:1, 150:1, 180:1, 200 :1, 230:1, 250:1, 280:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1 , 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, 1100:1, 1200:1, 1500:1, 2000:1, 2500:1, 3000:1, 3500 :1, 4000:1, 4500:1, 5000:1, 5500:1, 6000:1, 6500:1, 7000:1, 7500:1, 8000:1, 8500:1, 9000:1, 9500:1 Or 10000:1, preferably (100-1000):1, more preferably 100:1.
本发明中，通过加入所述DNB加载缓冲液，其拥有与RCA产物不一样的折光系数，保证待测样品RCA产物能够均匀的分散在液体介质中，做无规则地布朗运动，显著的提高了检测的准确性，发明人发现，为了减少其他粒子对待测样品产生影响，通过控制所述DNB加载缓冲液和所述DNB与所述PBS的体积比，将所述DNB进行稀释，可以进一步提高检测的准确性，减少其他自理对待测样品产生的影响，从而能够更精准的检测和区分DNB。In the present invention, by adding the DNB loading buffer, it has a different refractive index from that of the RCA product, ensuring that the RCA product of the sample to be tested can be uniformly dispersed in the liquid medium and perform random Brownian motion, which significantly improves the The detection accuracy, the inventors found that in order to reduce the impact of other particles on the sample to be tested, by controlling the DNB loading buffer and the volume ratio of the DNB to the PBS, and diluting the DNB, the detection can be further improved. It can reduce the influence of other self-care samples to be tested, so as to detect and distinguish DNB more accurately.
作为优选技术方案，所述检测RCA产物的方法，包括如下步骤：As a preferred technical scheme, the method for the described detection RCA product comprises the steps:
(1)制备环状DNA分子；(1) prepare circular DNA molecules;
(2)以环状DNA分子为模板，滚环扩增5-60min，获得RCA产物，加入荧光分子修饰的探针0.5-5μM，与所述RCA产物杂交，获得杂交产物；(2) using a circular DNA molecule as a template, performing rolling circle amplification for 5-60 min to obtain an RCA product, adding a probe modified by a fluorescent molecule at 0.5-5 μM, and hybridizing with the RCA product to obtain a hybrid product;
(3)将所述杂交产物与PBS缓冲液以体积比1:(9-999)混合得到混合液，再向所述混合液中加入与所述混合液体积比为(100-10000):1的加载缓冲液；(3) the hybridization product is mixed with the PBS buffer at a volume ratio of 1:(9-999) to obtain a mixed solution, and then the mixed solution is added to the mixed solution in a volume ratio of (100-10000):1 of loading buffer;
(4)采用粒径分析仪对所述杂交产物进行检测，获得粒径分析结果。(4) using a particle size analyzer to detect the hybridization product to obtain particle size analysis results.
另一方面，本发明提供一种DNA聚合酶的筛选方法，包括如下步骤：On the other hand, the present invention provides a method for screening DNA polymerase, comprising the steps of:
(1’)以所述环状DNA分子为模板，采用不同的DNA聚合酶进行滚环扩增，获得RCA产物；(1') using the circular DNA molecule as a template, using different DNA polymerases to carry out rolling circle amplification to obtain an RCA product;
(2’)采用粒径分析仪对所述RCA产物进行检测，获得粒径分析结果，进而筛选合适的DNA聚合酶；(2') adopt particle size analyzer to detect described RCA product, obtain particle size analysis result, and then screen suitable DNA polymerase;
其中，所述DNA聚合酶具有链置换活性。Wherein, the DNA polymerase has strand displacement activity.
本发明中，通过对不同大小的DNB的分析，可以准确地筛选出针对同一个模板的DNA聚合酶在相同的反应条件下可以聚合出多大的DNB，再根据实验目的需要的DNB大小，选用合适的DNA聚合酶。In the present invention, by analyzing DNBs of different sizes, it is possible to accurately screen out the size of DNBs that can be polymerized by DNA polymerase for the same template under the same reaction conditions. DNA polymerase.
根据本发明，步骤(1’)所述不同的DNA聚合酶由于其与模板的结合不同，具有不一样的扩增效率，从而制备得到不同大小的DNB，本领域技术人员可以根据需要进行制备，在此不做特殊限定。According to the present invention, the different DNA polymerases described in step (1') have different amplification efficiencies due to their different binding to the template, thereby preparing DNBs of different sizes, and those skilled in the art can prepare as needed, No special limitation is made here.
根据本发明，步骤(1’)所述的DNA聚合酶只要是能够进行RCA的DNA聚合酶都可行，选择越多的DNA聚合酶，筛选DNA聚合酶的范围越大，本领域技术人员可以根据需要进行选择，本发明中所选的DNA聚合酶为phi29DNA聚合酶，所述DNA聚合酶包括野生型DNA聚合酶和/或突变型DNA聚合酶。According to the present invention, the DNA polymerase described in step (1') is feasible as long as it is a DNA polymerase capable of performing RCA. It needs to be selected, the DNA polymerase selected in the present invention is phi29 DNA polymerase, and the DNA polymerase includes wild-type DNA polymerase and/or mutant DNA polymerase.
在一个具体的实施例中，步骤(1’)具体包括：取环化后的DNA文库20μL到多个PCR管内，加入20μL DNB制备缓冲液，漩涡振荡器震荡混匀，离心机离心后置于PCR仪中反应，反应条件为：95℃1min，65℃1min，40℃1min，4℃保持；当温度达到4℃时取出PCR管，离心机离心后，各反应管中均加入40μLDNA聚合酶反应混合液和4μL不同突变型的phi29聚合酶，漩涡振荡器震荡混匀，离心机离心后即刻置于PCR仪中开始反应，反应条件：30℃20min，4℃保持；反应结束后，加入20μL终止缓冲液，即获得RCA产物。In a specific embodiment, step (1') specifically includes: taking 20 μL of the circularized DNA library into multiple PCR tubes, adding 20 μL DNB preparation buffer, shaking and mixing with a vortex shaker, centrifuging in a centrifuge and placing The reaction conditions were as follows: 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, and hold at 4°C; when the temperature reached 4°C, take out the PCR tube, centrifuge in a centrifuge, and add 40 μL of DNA polymerase to each reaction tube to react The mixed solution and 4 μL of different mutant phi29 polymerases were shaken and mixed by a vortex shaker. Immediately after centrifugation, the reaction was started in the PCR instrument. The reaction conditions were: 30 °C for 20 min and kept at 4 °C; after the reaction, 20 μL was added to stop the reaction. buffer to obtain the RCA product.
根据本发明，步骤(1’)所述滚环扩增的时间为5-60min，例如可以是5min、10min、20min、30min、40min、50min或60min，通过采用不同的DNA聚合酶进行相同时间的滚环扩增，从而通过不同的RCA产物筛选DNA聚合酶。According to the present invention, the rolling circle amplification time of step (1') is 5-60min, for example, it can be 5min, 10min, 20min, 30min, 40min, 50min or 60min, by using different DNA polymerases for the same time Rolling circle amplification to screen DNA polymerases by different RCA products.
根据本发明，在步骤(1’)所述滚环扩增之后，还包括加入荧光分子修饰的探针与所述RCA产物杂交的步骤；According to the present invention, after the rolling circle amplification in step (1'), the step of adding a probe modified by a fluorescent molecule to hybridize with the RCA product is further included;
优选地，所述荧光分子为ROX、FAM、HEX、VIC、Cy5或Cy3中的任意一种或至少两种的组合，优选为ROX。Preferably, the fluorescent molecule is any one or a combination of at least two of ROX, FAM, HEX, VIC, Cy5 or Cy3, preferably ROX.
根据本发明，所述荧光分子修饰的探针的加入量为0.5-5μM，例如可以是0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1μM、1.1μM、1.2μM、1.3μM、1.5μM、1.8μM、2μM、2.3μM、2.5μM、2.8μM、3μM、3.2μM、3.5μM、3.8μM、4μM、4.2μM、4.5μM、4.8μM或5μM，优选为1μM。According to the present invention, the added amount of the fluorescent molecule-modified probe is 0.5-5 μM, for example, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.5 μM μM, 1.8 μM, 2 μM, 2.3 μM, 2.5 μM, 2.8 μM, 3 μM, 3.2 μM, 3.5 μM, 3.8 μM, 4 μM, 4.2 μM, 4.5 μM, 4.8 μM or 5 μM, preferably 1 μM.
根据本发明，所述RCA产物为DNA纳米球(DNA nanoball,DNB)，是使用RCA技术将单链环状DNA扩增多个数量级所产生的扩增产物。滚环扩增反应时间的不同会导致产生不同分子量大小的DNB。According to the present invention, the RCA product is a DNA nanoball (DNA nanoball, DNB), which is an amplification product produced by amplifying single-stranded circular DNA by multiple orders of magnitude using RCA technology. Different reaction times of rolling circle amplification will result in the generation of DNBs of different molecular weights.
根据本发明，所述粒径分析结果包括RCA产物的分子大小、RCA产物的分子分布或RCA产物的均一性中的任意一种或至少两种的组合。According to the present invention, the particle size analysis result includes any one or a combination of at least two of the molecular size of the RCA product, the molecular distribution of the RCA product, or the homogeneity of the RCA product.
根据本发明，步骤(1’)所述环状DNA文库的制备方法为本领域的常规方法，本领域技术人员可以根据需要进行选择，在此不做特殊限定。According to the present invention, the preparation method of the circular DNA library described in step (1') is a conventional method in the art, and those skilled in the art can select according to needs, which is not particularly limited here.
根据被本发明，在步骤(2)之前，还包括将步骤(1)获得的RCA产物进行稀释的步骤。According to the present invention, before step (2), the step of diluting the RCA product obtained in step (1) is further included.
根据被本发明，采用稀释液进行稀释，所述稀释液为PBS和/或加载缓冲液，优选为PBS和加载缓冲液的组合。According to the present invention, the dilution is performed using a diluent, the diluent being PBS and/or loading buffer, preferably a combination of PBS and loading buffer.
根据被本发明，所述加载缓冲液为非离子表面活性剂，优选为聚氧乙烯聚氧丙烯嵌段共聚物，进一步优选为泊洛沙姆188。According to the present invention, the loading buffer is a nonionic surfactant, preferably a polyoxyethylene polyoxypropylene block copolymer, more preferably poloxamer 188.
根据本发明，所述稀释具体包括：将RCA产物与PBS缓冲液混合得到混合液，再向所述混合液中加入所述加载缓冲液。According to the present invention, the dilution specifically includes: mixing the RCA product with a PBS buffer to obtain a mixed solution, and then adding the loading buffer to the mixed solution.
根据本发明，所述RCA产物与所述PBS缓冲液的体积比为1:(9-9999)，例如可以是1:9、1:10、1:12、1:15、1:20、1:30、1:40、1:50、1:60、1:70、1:80、1:90、1:99、1:100、1:120、1:150、1:180、1:200、1:250、1:300、1:350、1:400、450、1:480、1:499、1:500、1:600、1:700、1:800、1:900、1:950、1:999、1:1000、1:1100、1:1200、1:1500、1:1800、1:2000、1:2500、1:3000、1:3500、1:4000、1:4500、1:5000、1:5500、1:6000、1:6500、1:7000、1:7500、1:8000、1:8500、1:9000、1:9500或1:9999，优选为1:(99-999)，进一步优选为1:99。According to the present invention, the volume ratio of the RCA product to the PBS buffer is 1:(9-9999), such as 1:9, 1:10, 1:12, 1:15, 1:20, 1 :30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:99, 1:100, 1:120, 1:150, 1:180, 1:200 , 1:250, 1:300, 1:350, 1:400, 450, 1:480, 1:499, 1:500, 1:600, 1:700, 1:800, 1:900, 1:950 , 1:999, 1:1000, 1:1100, 1:1200, 1:1500, 1:1800, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1 :5000, 1:5500, 1:6000, 1:6500, 1:7000, 1:7500, 1:8000, 1:8500, 1:9000, 1:9500 or 1:9999, preferably 1:(99- 999), more preferably 1:99.
根据本发明，所述混合液与所述加载缓冲液的体积比为(100-10000):1，例如可以是100:1、110:1、120:1、150:1、180:1、200:1、230:1、250:1、280:1、300:1、350:1、400:1、450:1、500:1、550:1、600:1、650:1、700:1、750:1、800:1、850:1、900:1、950:1、1000:1、1100:1、1200:1、1500:1、2000:1、2500:1、3000:1、3500:1、4000:1、4500:1、5000:1、5500:1、6000:1、6500:1、7000:1、7500:1、8000:1、8500:1、9000:1、9500:1或10000:1，优选为(100-1000):1，进一步优选为100:1。According to the present invention, the volume ratio of the mixed solution to the loading buffer is (100-10000):1, for example, 100:1, 110:1, 120:1, 150:1, 180:1, 200 :1, 230:1, 250:1, 280:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1 , 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, 1100:1, 1200:1, 1500:1, 2000:1, 2500:1, 3000:1, 3500 :1, 4000:1, 4500:1, 5000:1, 5500:1, 6000:1, 6500:1, 7000:1, 7500:1, 8000:1, 8500:1, 9000:1, 9500:1 Or 10000:1, preferably (100-1000):1, more preferably 100:1.
本发明中，通过加入所述DNB加载缓冲液，其拥有与RCA产物不一样的折光系数，保证待测样品RCA产物能够均匀的分散在液体介质中，做无规则地布朗运动，显著的提高了检测的准确性，发明人发现，为了减少其他粒子对待测样品产生影响，通过控制所述DNB加载缓冲液和所述DNB与所述PBS的体积比，将所述DNB进行稀释，可以进一步提高检测的准确性，减少其他自理对待测样品产生的影响，从而能够更精准的检测和区分DNB。In the present invention, by adding the DNB loading buffer, it has a different refractive index from that of the RCA product, ensuring that the RCA product of the sample to be tested can be uniformly dispersed in the liquid medium and perform random Brownian motion, which significantly improves the The detection accuracy, the inventors found that in order to reduce the impact of other particles on the sample to be tested, by controlling the DNB loading buffer and the volume ratio of the DNB to the PBS, and diluting the DNB, the detection can be further improved. It can reduce the influence of other self-care samples to be tested, so as to detect and distinguish DNB more accurately.
作为优选技术方案，所述phi29聚合酶的筛选方法，包括如下步骤：As a preferred technical scheme, the screening method of the phi29 polymerase comprises the following steps:
(1’)制备环状DNA分子；(1') prepare a circular DNA molecule;
(2’)以环状DNA分子为模板，采用不同的DNA聚合酶进行滚环扩增5-60min，获得RCA产物，加入荧光分子修饰的探针0.5-5μM，与所述RCA产物杂交，获得杂交产物；(2') using a circular DNA molecule as a template, using different DNA polymerases to carry out rolling circle amplification for 5-60 min to obtain an RCA product, adding 0.5-5 μM of a fluorescent molecule-modified probe, and hybridizing with the RCA product to obtain hybrid product;
(3’)将所述杂交产物与PBS缓冲液以体积比1:(99-999)混合得到混合液，再向所述混合液中加入与所述混合液体积比为(100-1000):1的加载缓冲液；(3') the hybridization product and the PBS buffer are mixed with a volume ratio of 1:(99-999) to obtain a mixed solution, and then in the mixed solution, the volume ratio with the mixed solution is added to be (100-1000): 1 of loading buffer;
(4’)采用粒径分析仪对所述杂交产物进行检测，获得粒径分析结果，进而筛选合适的DNA聚合酶。(4') using a particle size analyzer to detect the hybridization product to obtain a particle size analysis result, and then screen a suitable DNA polymerase.
与现有技术相比，本发明至少具有以下有益效果：Compared with the prior art, the present invention at least has the following beneficial effects:
(1)本发明方法可以直接用于分析RCA产物的粒径，所述方法具有快速、准确且操作简便等优点，所述方法可用于优化RCA体系中的组分，从而提高RCA产物的均一性；(1) The method of the present invention can be directly used to analyze the particle size of the RCA product, the method has the advantages of being fast, accurate and easy to operate, and the method can be used to optimize the components in the RCA system, thereby improving the homogeneity of the RCA product ;
(2)本发明方法可以用来高通量筛选针对不同DNA模板所适合的DNA聚合酶的突变体，结合二代测序手段，可以提高合成的效率。(2) The method of the present invention can be used for high-throughput screening of mutants of DNA polymerases suitable for different DNA templates, and combined with next-generation sequencing methods, the synthesis efficiency can be improved.
附图说明Description of drawings
图1为突变型phi29聚合酶制备的DNB与野生型phi29聚合酶制备的DNB的粒径分布结果，其中,Median line为中位线，Mean为平均值，Outliers为离群值；Fig. 1 is the particle size distribution result of DNB prepared by mutant phi29 polymerase and DNB prepared by wild-type phi29 polymerase, wherein, Median line is median line, Mean is mean value, Outliers is outlier;
图2(A)野生型phi29聚合酶制备的DNB的信号分布结果，图2(B)为突变型phi29聚合酶制备的DNB的信号分布结果；Fig. 2(A) is the signal distribution result of DNB prepared by wild-type phi29 polymerase, and Fig. 2(B) is the signal distribution result of DNB prepared by mutant phi29 polymerase;
图3为突变型phi29聚合酶制备的DNB与野生型phi29聚合酶制备的DNB的Q30、转载效率和有效DNB比例的结果图。FIG. 3 is a graph showing the results of Q30, transfer efficiency and effective DNB ratio of DNB prepared by mutant phi29 polymerase and DNB prepared by wild type phi29 polymerase.
具体实施方式Detailed ways
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。In order to facilitate the understanding of the present invention, examples of the present invention are as follows. It should be understood by those skilled in the art that the embodiments are only for helping the understanding of the present invention, and should not be regarded as a specific limitation of the present invention.
以下实施例所用的试剂：Reagents used in the following examples:
1、SE50试剂盒：来源于华大基因的测序仪BGI-SEQ500RS配套使用的建库试剂盒以及单端上机测序试剂盒。1. SE50 kit: library construction kit and single-end sequencing kit used with BGI-SEQ500RS sequencer from BGI.
2、10％泊洛沙姆188：商品名为普兰尼克F68，购自sigma，货号P5556。2. 10% Poloxamer 188: the trade name is Pluronic F68, which is purchased from sigma, item number P5556.
3、1XPBS：购自Thermo Fisher，pH7.4。3. 1XPBS: purchased from Thermo Fisher, pH 7.4.
实施例1：RCA产物的检测方法Embodiment 1: the detection method of RCA product
(1)环化模板DNA序列制备：依照BGI-SEQ500RS平台的SE50试剂盒中的建库试剂盒以及试剂盒说明书提供的方法进行环化模板DNA的制备。具体如下：使用试剂盒，提取大肠杆菌的基因组，通过物理打断的方式将基因组DNA打断，跑DNA胶，回收片段大小为150-250bp的DNA片段，制成混合文库。取160ng DNA混合文库，用分子级水将体积补充至48μL，充分混匀后短暂离心，置于PCR仪上95℃孵育5min，孵育完毕即刻取出PCR管放置在冰上冷却，上述PCR管加入11.6μL 1μM环化序列(来源于SE50试剂盒)，以及0.5μL连接酶，充分混匀，短暂离心，37℃孵育30min；(1) Preparation of circularized template DNA sequence: The circularized template DNA was prepared according to the library building kit in the SE50 kit of the BGI-SEQ500RS platform and the method provided in the kit instructions. The details are as follows: using the kit, extract the genome of Escherichia coli, interrupt the genomic DNA by physical interruption, run DNA gel, and recover DNA fragments with a fragment size of 150-250bp to make a mixed library. Take 160ng of DNA mixed library, supplement the volume to 48μL with molecular-grade water, mix well, centrifuge briefly, and incubate at 95°C for 5min on the PCR machine. Immediately after the incubation, take out the PCR tube and place it on ice to cool. Add 11.6 to the above PCR tube. μL 1μM circularization sequence (from SE50 kit), and 0.5μL ligase, mix well, centrifuge briefly, and incubate at 37°C for 30min;
(2)不同反应时间DNB制备：分别取环化后的DNA文库20μL到三个0.2mL PCR管内，分别加入20μL DNB制备缓冲液(来源于SE50试剂盒中的DNB制备试剂盒)，漩涡振荡器震荡混匀，迷你离心机离心5s置于PCR仪中反应，反应条件为：95℃1min，65℃1min，40℃1min，4℃保持；当温度达到4℃时取出PCR管，迷你离心机离心5s后加入40μL DNB聚合酶反应液I和4μLDNB聚合酶II(来源于SE50试剂盒中的DNB制备试剂盒)，漩涡振荡器震荡混匀，迷你离心机离心5s后即刻置于PCR仪中开始反应，反应条件：30℃5min/20min/30min(总共设置3组：A组反应5min，B组反应20min，C组反应30min)，4℃保持；5min DNB反应结束后，加入20μL终止缓冲液(0.5M EDTA)，轻轻混匀；20min DNB反应结束后，同样加入20μL终止缓冲液(0.5MEDTA)，轻轻混匀终止反应；30min DNB反应结束后，同样加入20μL终止缓冲液(0.5M EDTA)，轻轻混匀终止反应；为保证待测样品的准确检测，用PBS和10％泊洛沙姆188稀释样品；在5minDNB(A组)、20min DNB(B组)及30min DNB(C组)中各取100μL，然后分别加入9900μL PBS及100μL 10％泊洛沙姆188，轻轻混匀；在制备好的三种DNB溶液中，均加入1μM带荧光修饰的探针(探针序列：5’-ROX GCTCACAGAACGACATGGCTACGATCCGACTT-3’(SEQ ID NO.1))，与DNB互补配对杂交，室温下孵育30min，为了验证重复性，每组反应重复两次；(2) DNB preparation at different reaction times: Take 20 μL of the circularized DNA library into three 0.2 mL PCR tubes, add 20 μL of DNB preparation buffer (from the DNB preparation kit in the SE50 kit), and vortex shaker Shake and mix well, centrifuge in a mini centrifuge for 5 s and place it in a PCR instrument to react. The reaction conditions are: 95 °C for 1 min, 65 °C for 1 min, 40 °C for 1 min, and hold at 4 °C; when the temperature reaches 4 °C, take out the PCR tube and centrifuge in the mini centrifuge. After 5s, 40μL of DNB polymerase reaction solution I and 4μL of DNB polymerase II (from the DNB preparation kit in the SE50 kit) were added, vortexed and mixed, and centrifuged in a mini centrifuge for 5s and placed in the PCR machine to start the reaction. , reaction conditions: 30°C for 5min/20min/30min (a total of 3 groups were set: 5min in group A, 20min in group B, and 30min in group C), kept at 4°C; after the 5min DNB reaction, 20 μL of stop buffer (0.5 M EDTA), mix gently; after the 20min DNB reaction, add 20μL of stop buffer (0.5MEDTA), mix gently to stop the reaction; after the 30min DNB reaction, add 20μL of stop buffer (0.5M EDTA) , gently mix to terminate the reaction; in order to ensure the accurate detection of the sample to be tested, dilute the sample with PBS and 10% poloxamer 188; in 5min DNB (group A), 20min DNB (group B) and 30min DNB (group C) Take 100 μL of each DNB solution, then add 9900 μL of PBS and 100 μL of 10% poloxamer 188, and mix gently; to the three prepared DNB solutions, add 1 μM of the fluorescently modified probe (probe sequence: 5 '-ROX GCTCACAGAACGACATGGCTACGATCCGACTT-3' (SEQ ID NO. 1)), hybridized with DNB, and incubated at room temperature for 30 min. To verify the repeatability, each group of reactions was repeated twice;
(3)纳米粒径分析仪检测：打开粒径分析仪(马尔文NanoSight NS300)，预热30min，分别将杂交后的5min DNB，20min DNB，30min DNB混合液依次加入到检测仪中进行检测，对所得的粒径分布图中的峰强，峰面积及粒径分布范围进行数据分析，结果如下表1所示。(3) Nanoparticle size analyzer detection: turn on the particle size analyzer (Malvern NanoSight NS300), preheat for 30min, respectively add the hybridized 5min DNB, 20min DNB, and 30min DNB mixture into the detector for detection, respectively, Data analysis was performed on the peak intensity, peak area and particle size distribution range in the obtained particle size distribution diagram, and the results are shown in Table 1 below.
表1Table 1
D10:样品中10％粒子直径小于该数值D10: 10% of the particles in the sample have a diameter smaller than this value
D50:样品中50％粒子直径小于该数值D50: 50% of the particles in the sample have a diameter smaller than this value
D90:样品中90％粒子直径小于该数值D90: 90% of the particles in the sample have a diameter smaller than this value
通过表1中不同时间的DNB平均粒径大小可以看出，30min DNB大于20min DNB大于5min DNB，这表明此粒径分析仪能够准确区分不同大小的DNB。从不同样品的粒径分布D10，D50以及D90的粒径大小看出，DNB在滚环复制20min后，粒径的增长速率要明显小于前20min的增长速率，这表明phi29DNA聚合酶在20min后的扩增效率已经开始下降。另外，从DNB浓度来看，RCA时间越长，粒子数越多，然而从粒径标准差可看出，RCA时间越长，粒径分布越不均匀。这些信息都有利于指导后续DNB制备的体系优化，制备出更加均匀的DNB，进一步提高测序质量。From the average particle size of DNB at different times in Table 1, it can be seen that 30min DNB is greater than 20min DNB is greater than 5min DNB, which indicates that this particle size analyzer can accurately distinguish DNBs of different sizes. From the particle size distribution D10, D50 and D90 of different samples, it can be seen that the growth rate of DNB particle size after 20min of rolling circle replication is significantly smaller than the growth rate of the first 20min, which indicates that phi29 DNA polymerase after 20min. Amplification efficiency has begun to decline. In addition, from the perspective of DNB concentration, the longer the RCA time, the more the number of particles, but from the standard deviation of the particle size, it can be seen that the longer the RCA time, the more uneven the particle size distribution. These information are helpful to guide the system optimization of subsequent DNB preparation, prepare more uniform DNB, and further improve the sequencing quality.
实施例2筛选phi29聚合酶Example 2 Screening of phi29 polymerase
(1)参考实施例1的步骤(1)制备混合文库；(1) with reference to step (1) of Example 1 to prepare a mixed library;
(2)采用不同phi29聚合酶制备不同DNB溶液：分别取环化后的DNA文库20μL到两个0.2mL PCR管内，分别加入20μL DNB制备缓冲液(来源于SE50试剂盒中的DNB制备试剂盒)漩涡振荡器震荡混匀，迷你离心机离心5s置于PCR仪中反应，反应条件为：95℃1min，65℃1min，40℃1min，4℃保持；当温度达到4℃时取出PCR管，迷你离心机离心5s后加入40μL DNA聚合酶反应液I(来源于SE50试剂盒中的DNB制备试剂盒)和4μL不同的phi29聚合酶(即野生型phi29聚合酶和待筛选的突变型phi29聚合酶)，漩涡振荡器震荡混匀，迷你离心机离心5s后即刻置于PCR仪中开始反应，反应条件：30℃20min，4℃保持；20min DNB反应结束后，加入20μL终止缓冲液(0.5M EDTA)，轻轻混匀，待用；(2) Use different phi29 polymerases to prepare different DNB solutions: respectively take 20 μL of the circularized DNA library into two 0.2 mL PCR tubes, and add 20 μL of DNB preparation buffer (from the DNB preparation kit in the SE50 kit) Vortex shaker to mix evenly, centrifuge for 5 s in a mini centrifuge and place it in a PCR instrument to react. The reaction conditions are: 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, and hold at 4°C; when the temperature reaches 4°C, take out the PCR tube, and the mini After centrifugation for 5 s, add 40 μL of DNA polymerase reaction solution I (derived from the DNB preparation kit in the SE50 kit) and 4 μL of different phi29 polymerases (ie wild-type phi29 polymerase and mutant phi29 polymerase to be screened) , vortex shaker to mix evenly, centrifuge in a mini centrifuge for 5 s and place it in the PCR instrument to start the reaction. The reaction conditions are: 30 °C for 20 min, keep at 4 °C; after the 20 min DNB reaction, add 20 μL of stop buffer (0.5M EDTA) , gently mix and set aside;
(3)向反应后的DNB加入9900μL PBS和100μL 10％泊洛沙姆188，将不同聚合酶反应产生的DNB溶液稀释至合适的浓度；然后，再加入1μM带荧光修饰的探针(探针序列：5’-ROX GCTCACAGAACGACATGGCTACGATCCGACTT-3’(SEQ ID NO.1))，与DNB互补配对杂交，室温下孵育30min；(3) Add 9900 μL of PBS and 100 μL of 10% poloxamer 188 to the reacted DNB, and dilute the DNB solutions generated by different polymerase reactions to an appropriate concentration; then, add 1 μM of fluorescently modified probe (probe Sequence: 5'-ROX GCTCCAGAACGACATGGCTACGATCCGACTT-3' (SEQ ID NO. 1)), hybridize with DNB, and incubate at room temperature for 30min;
(4)分别取2μL上述杂交后的DNB溶液进行Qubit定量，结果为野生型phi29聚合酶所在的DNB溶液浓度为27.3ng/μL，待筛选的突变型phi29聚合酶所在的DNB溶液浓度为25ng/μL；(4) Take 2 μL of the above hybridized DNB solution for Qubit quantification. The result is that the concentration of the DNB solution containing the wild-type phi29 polymerase is 27.3 ng/μL, and the concentration of the DNB solution containing the mutant phi29 polymerase to be screened is 25 ng/μL. μL;
(5)纳米粒径分析仪检测，采用粒径分析仪进行DNB的检测，通过得到的粒径分布图来区分不同大小的DNB，从而筛选合适的phi29聚合酶，结果如图1所示；(6)将步骤(2)中的DNB，按照BGISEQ500的DNB装载方法，将DNB分别装载于同批次的芯片上，在BGI-seq500上进行1个循环的测序检测，BGI-seq500的数据分析软件得到所示的不同信号分布及DNB在芯片上的加载情况，结果如图2(A)-图2(B)和图3所示。(5) Nano particle size analyzer detects, adopts particle size analyzer to detect DNB, and distinguishes DNB of different sizes through the obtained particle size distribution diagram, thereby screening suitable phi29 polymerase, the results are shown in Figure 1; ( 6) The DNB in step (2) is loaded on the same batch of chips according to the DNB loading method of BGISEQ500, and a cycle of sequencing detection is performed on the BGI-seq500. The data analysis software of BGI-seq500 The different signal distributions shown and the loading of DNB on the chip are obtained, and the results are shown in Figure 2(A)-Figure 2(B) and Figure 3.
从图1可以看出，从10％～90％及1％～99％的DNB分布来看，突变型聚合酶制备的DNB分布都显示比野生型的更加集中；此外，从离群点也能看出，使用突变型聚合酶制备的DNB，离群点偏离平均值及中位值数都低于野生型的。整体而言，突变型聚合酶制备的DNB更均匀。As can be seen from Figure 1, from the DNB distributions of 10% to 90% and 1% to 99%, the distribution of DNB prepared by mutant polymerase is more concentrated than that of wild type; It can be seen that the DNB prepared using the mutant polymerase has a lower number of outliers than the wild type in both mean and median deviations. Overall, the mutant polymerase produced more uniform DNB.
从图2(A)-图2(B)可以看出，突变型phi29聚合酶制备的DNB信号分布更均匀，而野生型phi29聚合酶制备的DNB较弥散。It can be seen from Figure 2(A)-Figure 2(B) that the signal distribution of DNB prepared by mutant phi29 polymerase is more uniform, while that of DNB prepared by wild type phi29 polymerase is more diffuse.
从图3可以看出，突变型phi29聚合酶制备的DNB较野生型phi29聚合酶制备的DNB，具有更高的装载效率，在装载到芯片上的DNB中，突变型phi29聚合酶制备的DNB能有效的进行后续测序的比例较大，且质量值(Q30)也高于野生型，意味着突变体DNB能够表现出更好的测序质量。As can be seen from Figure 3, DNB prepared by mutant phi29 polymerase has higher loading efficiency than DNB prepared by wild-type phi29 polymerase. Among the DNB loaded on the chip, DNB prepared by mutant phi29 polymerase can The proportion of effective subsequent sequencing is larger, and the quality value (Q30) is also higher than that of the wild type, which means that the mutant DNB can show better sequencing quality.
从上述结果说明，传统的qubit定量仪与上机测试的结果对应关系不大，即使qubit定量仪测试结果显示高浓度的DNB，并没有在测序仪上显示较高的信号值，而纳米粒径分析仪的结果与测序仪上的数据结果都显示突变体制备的DNB更加均匀，且能够获得更好测序质量，说明纳米粒径分析仪，能够快速有效地筛选出合适的phi29聚合酶。The above results show that the traditional qubit quantifier has little correspondence with the results of the on-board test. Even if the qubit quantifier test results show a high concentration of DNB, it does not show a high signal value on the sequencer, while the nanoparticle size The results of the analyzer and the data on the sequencer show that the DNB prepared by the mutant is more uniform and can obtain better sequencing quality, indicating that the nanoparticle size analyzer can quickly and effectively screen out the appropriate phi29 polymerase.
对比例使用DNB加载缓冲10％泊洛沙姆188Comparative Example Using
(1)环化模板DNA序列制备：具体制备步骤同实施例1的步骤(1)制备混合文库；(1) Preparation of circularized template DNA sequence: the specific preparation steps are the same as those in step (1) of Example 1 to prepare a mixed library;
(2)DNB制备：分别取环化后的DNA文库20μL到两个0.2mL PCR管内，分别加入20μLDNB制备缓冲液(来源于SE50试剂盒中的DNB制备试剂盒)漩涡振荡器震荡混匀，迷你离心机离心5s置于PCR仪中反应，反应条件为：95℃1min，65℃1min，40℃1min，4℃保持；当温度达到4℃时取出PCR管，迷你离心机离心5s后加入40μL DNA聚合酶反应液I(来源于SE50试剂盒中的DNB制备试剂盒)和4μLphi29聚合酶，漩涡振荡器震荡混匀，迷你离心机离心5s后即刻置于PCR仪中开始反应，反应条件：30℃20min，4℃保持；20min DNB反应结束后，加入20μL终止缓冲液(0.5M EDTA)，轻轻混匀。取50μL 20min DNB，加入5000μL PBS，轻轻混匀，标记为样品D；剩余50μL DNB加入4950μL PBS及50μL10％泊洛沙姆188，轻轻混匀，标记为样品E，为了验证重复性，对样品D和样品E进行两次重复试验；(2) DNB preparation: Take 20 μL of the circularized DNA library into two 0.2 mL PCR tubes, respectively, add 20 μL of DNB preparation buffer (derived from the DNB preparation kit in the SE50 kit) and mix with a vortex shaker. Centrifuge for 5 s and place it in a PCR machine for reaction. The reaction conditions are: 95 °C for 1 min, 65 °C for 1 min, 40 °C for 1 min, and hold at 4 °C; when the temperature reaches 4 °C, take out the PCR tube, centrifuge in a mini centrifuge for 5 s, and add 40 μL of DNA The polymerase reaction solution I (from the DNB preparation kit in the SE50 kit) and 4 μL of phi29 polymerase were mixed with a vortex shaker. After centrifugation in a mini centrifuge for 5s, the reaction was started immediately in the PCR machine. Reaction conditions: 30°C Keep at 4°C for 20 min; after the 20 min DNB reaction, add 20 μL of stop buffer (0.5M EDTA) and mix gently. Take 50 μL of 20min DNB, add 5000 μL of PBS, mix gently, and mark it as sample D; add 4950 μL of PBS and 50 μL of 10% poloxamer 188 to the remaining 50 μL of DNB, mix gently, and mark it as sample E. Sample D and sample E were tested twice;
(3)纳米粒径分析仪检测：打开粒径分析仪(马尔文NanoSight NS300)，预热30min，分别将样品D和样品E依次加入到检测仪中进行检测，对所得的粒径分布图中的峰强，峰面积及粒径分布范围进行数据分析，结果如表2所示。(3) Nano-particle size analyzer detection: turn on the particle size analyzer (Malvern NanoSight NS300), preheat for 30min, respectively add sample D and sample E into the detector for detection in turn. The peak intensity, peak area and particle size distribution range of the data were analyzed, and the results are shown in Table 2.
表2Table 2
D10：样品中10％粒子直径小于该数值D10: 10% of the particles in the sample have a diameter smaller than this value
D50：样品中50％粒子直径小于该数值D50: 50% of the particles in the sample have a diameter smaller than this value
D90：样品中90％粒子直径小于该数值D90: 90% of the particles in the sample have a diameter smaller than this value
从表2的实施例与对比例的结果可以看出，样品D1、D2由于没有加入本发明的加载缓冲液，两个样品的重复性较差，测出来的粒径大小差异较大，单位体积中的粒子数目差异也较大，而加入了本发明的加载缓冲液的样品E1、E2，数据重复性较好。As can be seen from the results of Examples and Comparative Examples in Table 2, since the loading buffer of the present invention was not added to Samples D1 and D2, the repeatability of the two samples was poor, and the measured particle size differences were large, and the unit volume The difference in the number of particles is also large, and the samples E1 and E2 added with the loading buffer of the present invention have better data repeatability.
综上所述，本发明方法可以直接用于分析RCA产物的粒径，所述方法具有快速、准确且操作简便等优点，所述方法可用于优化RCA体系中的组分，从而提高RCA产物的均一性；不仅如此，本发明方法还可以用来高通量筛选针对不同DNA模板所适合的DNA聚合酶的突变体，结合二代测序手段，可以显著提高合成的效率。To sum up, the method of the present invention can be directly used to analyze the particle size of RCA products, the method has the advantages of rapidity, accuracy and simple operation, and the method can be used to optimize the components in the RCA system, thereby improving the RCA products. Uniformity; not only that, the method of the present invention can also be used for high-throughput screening of DNA polymerase mutants suitable for different DNA templates, and combined with next-generation sequencing methods, the synthesis efficiency can be significantly improved.
申请人声明，本发明通过上述实施例来说明本发明的详细工艺设备和工艺流程，但本发明并不局限于上述详细工艺设备和工艺流程，即不意味着本发明必须依赖上述详细工艺设备和工艺流程才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。The applicant declares that the present invention illustrates the detailed process equipment and process flow of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, that is, it does not mean that the present invention must rely on the above-mentioned detailed process equipment and process flow. Process flow can be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
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