WO2016095736A1 - 一种基于多重pcr的目标区域富集方法和试剂 - Google Patents

一种基于多重pcr的目标区域富集方法和试剂 Download PDF

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WO2016095736A1
WO2016095736A1 PCT/CN2015/096790 CN2015096790W WO2016095736A1 WO 2016095736 A1 WO2016095736 A1 WO 2016095736A1 CN 2015096790 W CN2015096790 W CN 2015096790W WO 2016095736 A1 WO2016095736 A1 WO 2016095736A1
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primer
sequence
linker
target region
tag
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French (fr)
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郭晶
郭荣荣
李梅艳
耿春雨
蒋慧
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to US15/537,396 priority patent/US10435736B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

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  • the invention relates to the technical field of molecular biology, in particular to a target region enrichment method and reagent based on multiplex PCR.
  • Ampliseq is incapable of enriching the target region of cell-free DNA, because cell-free DNA has a small fragment size and its length is even smaller than the length of the Ampliseq kit PCR product.
  • Ampliseq-based region capture technology is only available. Only after PCR can the hybrid construction between samples be made, which will make a relatively time consuming process.
  • the invention provides a multiplex PCR-based target region enrichment method and reagent, which can realize region capture of short fragments of cell free DNA, and greatly reduce the time and cost of region capture.
  • the present invention provides a multiplex PCR-based target region enrichment method, comprising the steps of:
  • the first linker and the second linker are respectively ligated to the two ends of the nucleic acid fragment containing the target region to be enriched by a ligase to obtain a linker ligation product, wherein the first linker comprises a first linker fixed sequence for subsequent PCR amplification. a first tag sequence for labeling different samples and a second tag sequence for tagging molecular sequences of different target regions;
  • the first ligation product is subjected to PCR amplification using a first primer that specifically binds to the first linker fixing sequence and a second primer that specifically binds to the second linker to obtain an amplification product, wherein the first primer or the second primer
  • the primer has a first affinity tag
  • the third primer is used for linear amplification of the single primer, wherein the third primer comprises a third primer immobilization sequence at the 5' end and a target region-specific binding sequence at the 3' end;
  • the linear amplification product was used as a template, and the third primer and the first primer were used for exponential amplification to obtain a product containing the target region.
  • the first tag sequence is a sequence of 5-10 bases in length and the second tag sequence is a random sequence of 10-12 bases in length.
  • the nucleic acid fragment containing the target region to be enriched is a terminally repaired nucleic acid fragment.
  • the first primer has a first affinity tag.
  • the first affinity tag is a biotin tag
  • the second affinity tag is a streptavidin tag
  • the solid phase carrier is a magnetic bead
  • the invention provides a multiplex PCR-based target region enrichment reagent comprising the following components:
  • first linker comprises the first for subsequent PCR amplification a linker immobilization sequence, a first tag sequence for labeling different samples, and a second tag sequence for tagging molecular sequences of different target regions;
  • first primer and a second primer for PCR amplification of the linker ligation product to obtain an amplification product, wherein the first primer specifically binds to the first linker fixing sequence, the second primer specifically binds to the second linker, and the first The primer or the second primer has a first affinity tag;
  • a solid phase vector for capturing a single strand with a first affinity tag in the amplification product, wherein the solid phase carrier carries a second affinity tag capable of affinity binding to the first affinity tag;
  • a third primer for linear amplification of a single primer using the captured single strand as a template, wherein the third primer comprises a third primer immobilization sequence at the 5' end and a target region specific binding sequence at the 3' end;
  • the third primer and the first primer are also used for exponential amplification using a linear amplification product as a template to obtain a product containing a target region.
  • the first tag sequence is a sequence of 5-10 bases in length
  • the second standard The sequence is a random sequence of 10-12 bases in length.
  • the nucleic acid fragment containing the target region to be enriched is a terminally repaired nucleic acid fragment.
  • the first primer has a first affinity tag.
  • the first affinity tag is a biotin tag
  • the second affinity tag is a streptavidin tag
  • the solid phase carrier is a magnetic bead
  • the present invention is based on a multiplex PCR-based target region enrichment method.
  • the product linked to the tag sequence is linearly amplified and enriched in the target region, and then the target region is subjected to PCR enrichment through a specific primer (third primer), thereby making the multiple PCR is no longer limited by the effects of short-fragment nucleic acid sequences, enabling the capture of regions of short fragments of cell-free DNA; products linked to specific tag sequences (first tag sequences) can be achieved before enrichment of the target region.
  • Hybrid operation between multiple samples which greatly reduces the cost of sample processing and increases the throughput of sample processing.
  • the present invention uses high-weight molecular labels (second label sequences) to label molecules derived from different target regions within the same sample. Based on the high-weight molecular tag, the same molecule produced by PCR amplification can be identified, so that the molecular sequence and normal sequence of low mutation rate (such as one thousandth mutation rate) can be resolved under high-depth sequencing, so as to be low Mutated samples were screened and studied.
  • FIG. 1 is a schematic diagram showing the principle of an embodiment of a target region enrichment method based on multiplex PCR according to the present invention.
  • the main steps of the multiplex PCR-based target region enrichment method include: linker ligation; PCR amplification and biotin labeling; single strand selection; third primer binding; linear amplification; And exponential amplification. specifically:
  • Linker ligation ie, at the two ends of the nucleic acid fragment containing the target region to be enriched by the ligase Linking the first linker and the second linker to obtain a linker ligation product, wherein the first linker comprises a first linker set sequence for subsequent PCR amplification, a first tag sequence for labeling different samples, and a molecule for labeling different target regions The second tag sequence of the sequence.
  • the nucleic acid fragment may be a nucleic acid fragment containing a target region to be enriched from any source, and typical but non-limiting examples are: a random fragment obtained by cleavage of a genomic DNA by a physical disruption or a transposition enzyme-embedded complex, and PCR amplification.
  • a product of a product, a whole gemome amplification (WGA) product, or the like, particularly a short fragment molecule such as cell free DNA can also be used as a nucleic acid fragment of the present invention for enrichment of a target region.
  • the nucleic acid fragments produced by the above methods may require end-repair, especially the nucleic acid fragments generated by physical disruption, and need to be end-filled with DNA polymerase, and phosphorylated using DNA phosphorylation kinase, so that the subsequent ligation reaction proceeds smoothly. .
  • Both the first linker and the second linker are double-stranded short nucleotide sequences ranging in length from a dozen base pairs to tens of base pairs.
  • the first adaptor sequence is located at the 5' end of the sequence for subsequent PCR amplification, ie, as a primer annealing site for subsequent PCR amplification, the first tag sequence and the second tag sequence are located in the first linker.
  • Downstream (ie, 3' end) of the sequence, the order (or position) of the first tag sequence and the second tag sequence may not be limited, that is, the first tag sequence may be located upstream (ie, 5' end) of the second tag sequence or Downstream (ie 3' end) and vice versa.
  • the term "fixed sequence” means that the sequence of the segment is fixed, while other partial sequences can be changed. Therefore, the concept of "fixed sequence” can also be called “invariant sequence” or "universal sequence".
  • two adjacent sequences in the first linker fixing sequence, the first tag sequence and the second tag sequence may be directly connected, or may have a spacing of several bases, that is, connected by a "connection sequence” to form a first connector. .
  • the first tag sequence is used to label different samples, that is to say, the nucleic acid fragments of the same sample source are connected with the same first tag sequence after the linker is connected, and the nucleic acid fragments of different sample sources are connected with different numbers after being connected by the linker.
  • a sequence of tags so that the present invention does not allow for the mixing of different samples between the different regions after the target region is enriched by PCR, as in the conventional method, but can be performed between multiple samples before the target region is enriched.
  • the mixing operation greatly reduces the cost of sample processing and can increase the throughput of sample processing.
  • the second tag sequence is used to label the molecular sequences of different target regions, that is, the target region nucleic acid fragments in the same sample are connected with different second tag sequences after being connected by the linker. Since the second tag sequence is a random sequence, the length is generally 10- Between 12 bases, theoretically for a random sequence with N bases, the second tag sequence should have 4 N species, enough to mark more than one million target regions. Therefore, the high-weight-based molecular tag (second tag sequence) of the present invention can recognize whether the same molecule produced by PCR amplification can be identified, thereby distinguishing a low mutation rate (such as a thousandth mutation rate) under high-depth sequencing. Molecular and normal sequences to facilitate screening and study of low-mutation samples.
  • first tag sequence and the second tag sequence in the present invention does not refer to a sequence segment having a specific sequence, but a set of "tag sets" which vary based on the source of the sample and the source of the nucleic acid fragment molecules.
  • the corresponding first linker is also not a sequence segment with a particular sequence, but rather a set of "joint sets”.
  • the second linker is a sequence segment having a specific sequence which is versatile in the present invention for PCR amplification of the enriched target region.
  • the linker ligation step is to respectively connect the first linker and the second linker at both ends of the nucleic acid fragment containing the target region to be enriched, since the first linker and the second linker may be due to the inability to selectively connect, The occurrence of simultaneous insertion of two first linkers or two second linkers at the same nucleic acid fragment. Nevertheless, the effectiveness of the present invention is not affected because the first primer used in the PCR amplification step after the linker ligation specifically binds to the first linker fixing sequence, and the second primer specifically binds to the second linker, thereby Nucleic acid fragments connecting the first linker and the second linker, respectively, at the desired ends are enriched.
  • connection method may be: flat end connection or glue end connection, if the first joint and the first The two connectors are respectively connected by different methods, so that the same connector can be avoided at the two ends of the same nucleic acid fragment.
  • PCR amplification and biotin labeling that is, using a first primer that specifically binds to the first linker fixing sequence and a second primer that specifically binds to the second linker, PCR-amplification of the linker ligation product to obtain an amplification product, wherein A primer or second primer has a first affinity tag.
  • the first primer or the second primer is labeled with biotin as the first affinity tag; accordingly, the solid phase carrier is labeled with streptavidin as the second affinity tag.
  • the biotin-streptavidin system is the most widely used and most effective bioaffinity labeling method in biology. Of course, the invention is not limited thereto.
  • the first affinity tag it may be a component of a biologically common biological binding reaction, such as an antigen or antibody, a strand of a short-stranded DNA fragment, biotin or streptavidin, and the like.
  • the second affinity tag selects an antibody that binds to the antigen, and vice versa; in the first affinity tag, a double-stranded DNA short segment is used. In the case of one strand, the second affinity tag selects another strand that is complementary to the strand, and vice versa; in the case where biotin is selected for the first affinity tag, the second affinity tag is selected for binding to biotin. Streptavidin and vice versa.
  • the solid phase carrier is a magnetic bead, and as the solid phase carrier, a microplate, a chip or the like may be selected as long as the second affinity tag (such as streptavidin) can be immobilized to the corresponding solid.
  • the phase carrier is sufficient, and the magnetic beads are most preferred in the present invention.
  • Single strand selection i.e., capture of a single strand of the amplified product with a first affinity tag by a solid phase support with a second affinity tag capable of affinity binding to the first affinity tag.
  • the solid support is a magnetic bead
  • the first affinity tag is a biotin tag
  • the second affinity tag is a streptavidin tag. Therefore, the streptavidin-labeled magnetic beads are used to select a biotin-labeled single strand in the PCR amplification product by binding of streptavidin to biotin, and then used for downstream target region enrichment operations.
  • the third primer binds and linearly amplifies, that is, the single primer is used as a template, and the third primer is used for linear amplification of the single primer, wherein the third primer includes the third primer immobilization sequence at the 5' end and the target region specificity at the 3' end. Binding sequence.
  • the third primer fixed sequence means that the sequence is fixed, and other partial sequences can be changed, so the concept of "fixed sequence” can also be called “invariant sequence” or "universal sequence”.
  • the target region-specific binding sequence at the 3' end of the third primer is capable of specifically binding to the target region to be enriched, and then enrichment of the target region by PCR amplification.
  • the exponential amplification that is, the linear amplification product is used as a template, and the third primer and the first primer are used for exponential amplification to obtain a product containing the target region. At this point, the enrichment of the target area is achieved.
  • the enriched product of the target region can be applied to subsequent high throughput sequencing or other analytical platforms.
  • the method of the present invention can also be applied to other various tissue samples, even paraffin-embedded formaldehyde-fixed medical samples.
  • the underlined part of the first linker sequence is the first tag sequence for labeling different samples
  • the "N” part is the second tag sequence for labeling the molecular sequences of different target regions
  • the other part is for subsequent PCR amplification.
  • the first linker fixed sequence except for the three bases adjacent to the first tag sequence
  • 2 the underlined portion of the third primer sequence is the 3'-end target region-specific binding sequence, and the other portion is the 5' end of the third sequence
  • Primer immobilization sequence 3 all sequences are listed in 5' to 3' format.
  • peripheral blood tumor cell sample DNA (100 ng) is end-repaired and then ligated to the specific sequence of the linker (the first linker and the second linker) by blunt end or sticky end, and subjected to 5-7 cycles of amplification.
  • one primer (first primer) amplified by PCR carries a biotin modification or other label capable of specific strand separation.
  • the linearly amplified product is subjected to multiplex expansion.
  • the upstream primer of the amplification primer is a biotin-labeled strand-specific primer (first primer) in a 1.3-step PCR amplification system, and the downstream primer is linear amplification.
  • Primer set (third primer). The systems and procedures for multiple exponential amplification are shown in Tables 8 and 9 below:
  • the final multi-exponential amplified product is purified and subjected to sequencing analysis on the machine.
  • the analysis includes the enrichment ratio of the target area, coverage uniformity and variation. The results are shown in Table 10 below:
  • the total number of reads refers to the total number of reads read in the sequencing; the number of targets refers to the total number of reads read in the target area; the proportion of targets refers to the ratio of the total number of target areas to the total number of reads;
  • the target number coverage refers to the proportion of the target area coverage area; the target area capture efficiency refers to the proportion of the target area to the entire reference genome target area; the number of single base mutations refers to the target area detected The number of single base mutations.
  • the present application also provides another embodiment as shown below to demonstrate the feasibility of the method.
  • This example includes the same experimental procedures and materials as the above examples except that the lung cancer sample is substituted for the peripheral blood tumor cell sample. The results are shown in Table 11 below:
  • Table 12 shows the results of the allele frequencies obtained by the conventional hybridization capture library and the multiplex PCR-based target region enrichment method of the present invention at one gene position (chr7: 55242469).
  • Table 11 and Table 12 show that the multiplex PCR-based target region enrichment method of the present invention has a capture efficiency of up to 89.10% in the target region, and can detect the same results as the conventional hybrid capture capture library, and the frequencies are approximately similar and the results are similar. Consistently, it can be determined that the method of the present invention can be applied to target region enrichment.

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Abstract

提供一种基于多重PCR的目标区域富集方法和试剂，该方法包括：在含待富集目标区域的核酸片段两端分别连接第一接头和第二接头，得到接头连接产物；使用特异性结合第一接头的第一引物和特异性结合第二接头的第二引物，对接头连接产物进行PCR扩增，得到扩增产物，其中第一引物或第二引物具有第一亲和标记；通过固相载体捕获扩增产物中带有第一亲和标记的单链；以捕获的单链为模板，使用第三引物进行单引物线性扩增；以线性扩增产物为模板，使用第三引物和第一引物进行指数扩增，得到包含目标区域的产物。

Description

一种基于多重PCR的目标区域富集方法和试剂 技术领域
本发明涉及分子生物学技术领域，尤其涉及一种基于多重PCR的目标区域富集方法和试剂。
背景技术
随着DNA测序技术的发展，高通量测序技术已被广泛地应用于生命科学研究的各个领域。尽管随着测序技术的不断更新和普及，测序技术的成本也越来越低，但全基因组测序技术本身的花费还是很昂贵。用于调和这一问题的较好的方案是将感兴趣的目标区域富集出来，再进行高通量测序。传统的序列捕获技术，通常是将高通量测序文库构建好后，利用探针进行目标区域的文库富集再测序。
除此之外Life Tech公司基于多重PCR技术开发了一种称为Ampliseq的试剂盒，可以通过多重PCR过程实现目标区域的富集，从而大大缩减了杂交捕获这个耗时的操作过程，可以称为区域捕获技术的一项革新。
但是Ampliseq对于细胞游离DNA的目标区域富集却无能为力，因为细胞游离DNA具有片段小的特点，其长度甚至已经小于Ampliseq试剂盒PCR产物的长度；除此之外，基于Ampliseq的区域捕获技术只有在PCR后才能够实现样本之间的混合建库，这将使一个比较耗时的过程。
发明内容
本发明提供一种基于多重PCR的目标区域富集方法和试剂，能够实现细胞游离DNA这种短片段的区域捕获，并且大大减小区域捕获的时间和成本。
根据本发明的第一方面，本发明提供一种基于多重PCR的目标区域富集方法，包括如下步骤:
在连接酶作用下，在含待富集目标区域的核酸片段两端分别连接第一接头和第二接头，得到接头连接产物，其中第一接头包括用于后续PCR扩增的第一接头固定序列、用于标记不同样本的第一标签序列和用于标记不同目标区域分子序列的第二标签序列；
使用特异性结合第一接头固定序列的第一引物和特异性结合第二接头的第二引物，对接头连接产物进行PCR扩增，得到扩增产物，其中第一引物或第二 引物具有第一亲和标记；
通过固相载体捕获扩增产物中带有第一亲和标记的单链，其中固相载体上带有能够与第一亲和标记亲和结合的第二亲和标记；
以捕获的单链为模板，使用第三引物进行单引物线性扩增，其中第三引物包括5’端的第三引物固定序列和3’端的目标区域特异性结合序列；
以线性扩增产物为模板，使用第三引物和第一引物进行指数扩增，得到包含目标区域的产物。
作为本发明的优选方案，第一标签序列为5-10个碱基长度的序列，第二标签序列为10-12个碱基长度的随机序列。
作为本发明的优选方案，含待富集目标区域的核酸片段为末端修复过的核酸片段。
作为本发明的优选方案，第一引物具有第一亲和标记。
作为本发明的优选方案，第一亲和标记为生物素标记，第二亲和标记为链霉亲和素标记，固相载体为磁珠。
根据本发明的第二方面，本发明提供一种基于多重PCR的目标区域富集试剂，包括如下组成部分：
第一接头和第二接头，用于在连接酶作用下分别连接到含待富集目标区域的核酸片段两端，以得到接头连接产物，其中第一接头包括用于后续PCR扩增的第一接头固定序列、用于标记不同样本的第一标签序列和用于标记不同目标区域分子序列的第二标签序列；
第一引物和第二引物，用于对接头连接产物进行PCR扩增，得到扩增产物，其中第一引物特异性结合第一接头固定序列，第二引物特异性结合第二接头，并且第一引物或第二引物具有第一亲和标记；
固相载体，用于捕获扩增产物中带有第一亲和标记的单链，其中固相载体上带有能够与第一亲和标记亲和结合的第二亲和标记；
第三引物，用于以捕获的单链为模板，进行单引物线性扩增，其中第三引物包括5’端的第三引物固定序列和3’端的目标区域特异性结合序列；
其中，第三引物和第一引物还用于以线性扩增产物为模板，进行指数扩增，得到包含目标区域的产物。
作为本发明的优选方案，第一标签序列为5-10个碱基长度的序列，第二标 签序列为10-12个碱基长度的随机序列。
作为本发明的优选方案，含待富集目标区域的核酸片段为末端修复过的核酸片段。
作为本发明的优选方案，第一引物具有第一亲和标记。
作为本发明的优选方案，第一亲和标记为生物素标记，第二亲和标记为链霉亲和素标记，固相载体为磁珠。
本发明基于多重PCR的目标区域富集方法，首先将连接有标签序列的产物进行目标区域的线性扩增富集，然后通过特定引物(第三引物)将目标区域进行PCR富集，从而使得多重PCR不再受限于短片段核酸序列的影响，能实现细胞游离DNA这种短片段分子的区域捕获；连接有特定标签序列(第一标签序列)的产物可以在目标区域富集之前，就实现多样本之间的混合操作，这大大降低了样本处理的成本，并提高样本处理的通量。
此外，本发明除对不同样本间进行不同标签(第一标签序列)的标记以外，还使用高重数目分子标签(第二标签序列)对同一样本内不同目标区域来源的分子进行标记。基于高重数目分子标签可以对是否PCR扩增产生的相同分子进行识别，从而在高深度测序下可以分辨低突变率(如千分之一突变率)的分子序列和正常序列，以便于对低突变的样本进行筛查和研究。
附图说明
图1为本发明基于多重PCR的目标区域富集方法的一个实施方案的原理示意图。
具体实施方式
下面通过具体实施方式对本发明作进一步详细说明。除非特别说明，下面实施例中所使用的技术均为本领域内的技术人员已知的常规技术；所使用的仪器设备和试剂等，均为本领域内的技术人员可以通过公共途径如商购等获得的。
本发明中，任何情况下使用的“第一”、“第二”和“第三”等概念都不应当理解为具有顺序和技术的含义，其作用仅在于将其与其它对象区别开来。
请参考图1，本发明一个实施方案中，基于多重PCR的目标区域富集方法的主要步骤包括：接头连接；PCR扩增及生物素标记；单链选择；第三引物结合；线性扩增；和指数扩增。具体地：
接头连接，即在连接酶作用下，在含待富集目标区域的核酸片段两端分别 连接第一接头和第二接头，得到接头连接产物，其中第一接头包括用于后续PCR扩增的第一接头固定序列、用于标记不同样本的第一标签序列和用于标记不同目标区域分子序列的第二标签序列。
上述核酸片段可以是任何来源的含待富集目标区域的核酸片段，典型但非限定性的例子比如：基因组DNA经物理打断或转座酶包埋复合体打断得到的随机片段、PCR扩增产物、全基因组扩增(whole gemome amplification，WGA)产物等，特别是细胞游离DNA这种短片段分子也可作为本发明的核酸片段用于富集目标区域。
上述方法产生的核酸片段可能需要进行末端修复，尤其是物理打断产生的核酸片段，需要使用DNA聚合酶进行末端补平，使用DNA磷酸化激酶进行磷酸化处理，以便于后续接头连接反应顺利进行。
第一接头和第二接头均是双链短核苷酸序列，长度从十几个碱基对到几十个碱基对不等。
对于第一接头，其第一接头固定序列位于序列5’端，用于后续PCR扩增，即作为后续PCR扩增的引物退火位点，第一标签序列和第二标签序列位于第一接头固定序列的下游(即3’端)，至于第一标签序列和第二标签序列的顺序(或者位置)可以不限定，即第一标签序列可以位于第二标签序列的上游(即5’端)或下游(即3’端)，反之亦然。所谓“固定序列”，是指这一段序列是固定不变的，而其它部分序列可以变化，因此“固定序列”的概念也可以称作“不变序列”或“通用序列”等。此外，第一接头固定序列、第一标签序列和第二标签序列中两个相邻的序列可以直接相连，也可以有几个碱基的间隔，即通过“连接序列”连接起来形成第一接头。
第一标签序列用于标记不同样本，也就是说同一样本来源的核酸片段经接头连接以后会带上相同的第一标签序列，而不同样本来源的核酸片段经接头连接以后会带上不同的第一标签序列，从而使得本发明不像传统方法那样只有在通过PCR对目标区域富集后才能够进行不同样本之间的混合建库，而是可以在目标区域富集之前就进行多样本之间的混合操作，这大大降低了样本处理的成本，并可以提高样本处理的通量。
第二标签序列用于标记不同目标区域分子序列，即同一样本内的目标区域核酸片段经接头连接以后会带上不同的第二标签序列，由于第二标签序列是随 机序列，长度一般在10-12个碱基之间，理论上对于具有N个碱基的随机序列，第二标签序列的种类应该有4N种，足以标记数以百万计以上的目标区域。因此，本发明这种基于高重数目分子标签(第二标签序列)可以对是否PCR扩增产生的相同分子进行识别，从而在高深度测序下可以分辨低突变率(如千分之一突变率)的分子序列和正常序列，以便于对低突变的样本进行筛查和研究。
基于上述说明，本发明中第一标签序列、第二标签序列这样的概念并不是指一个具有特定序列的序列段，而是基于样本来源和核酸片段分子来源而有变化的一组“标签集”，相应的第一接头也并非一个具有特定序列的序列段，而是指一组“接头集”。然而，第二接头却是一个具有特定序列的序列段，在本发明中是通用的，用于PCR扩增富集目标区域。
虽然接头连接步骤的意图在于在含待富集目标区域的核酸片段两端分别连接第一接头和第二接头，但是由于第一接头和第二接头可能由于不具有选择性连接的能力，而导致两个第一接头或两个第二接头同时连接在同一个核酸片段两端的情况发生。尽管如此，也不影响本发明的有效性，因为接头连接以后的PCR扩增步骤中使用的第一引物特异性结合第一接头固定序列，而第二引物特异性结合第二接头，从而将那些期望的两端分别连接第一接头和第二接头的核酸片段富集起来。当然，也可以通过对连接方式的设计实现第一接头和第二接头分别特异性连接到核酸片段的两端，比如可以采用的连接方式有：平端连接或粘端连接，如果第一接头和第二接头分别采用不同的连接方式，即可避免同一核酸片段两端连接同一接头的情况发生。
PCR扩增及生物素标记，即使用特异性结合第一接头固定序列的第一引物和特异性结合第二接头的第二引物，对接头连接产物进行PCR扩增，得到扩增产物，其中第一引物或第二引物具有第一亲和标记。
本发明的一个实施方案中，以生物素作为第一亲和标记来标记第一引物或第二引物；相应地，以链霉亲和素作为第二亲和标记来标记固相载体。生物素-链霉亲和素系统是生物学中应用最广泛、效果最优异的生物亲和标记方法。当然，本发明并不局限于此。作为第一亲和标记，可以是生物学上常用的生物结合反应的一个组成部分，比如抗原或抗体，双链DNA短片段的一条链，生物素或链霉亲和素，等等。在第一亲和标记选用了抗原的情况下，第二亲和标记选用与该抗原结合的抗体，反之亦然；在第一亲和标记选用了双链DNA短片段的 一条链的情况下，第二亲和标记选用与该链互补配对的另一条链，反之亦然；在第一亲和标记选用了生物素的情况下，第二亲和标记选用与生物素结合的链霉亲和素，反之亦然。
本发明的一个实施方案中，固相载体是磁珠，作为固相载体还可以选用微孔板、芯片等，只要能将第二亲和标记(如链霉亲和素)固定到相应的固相载体即可，本发明最优选的是磁珠。
单链选择，即通过固相载体捕获扩增产物中带有第一亲和标记的单链，其中固相载体上带有能够与第一亲和标记亲和结合的第二亲和标记。
本发明的一个实施方案中，固相载体为磁珠，第一亲和标记为生物素标记，第二亲和标记为链霉亲和素标记。因此，使用链霉亲和素标记的磁珠通过链霉亲和素与生物素的结合，来选择PCR扩增产物中带有生物素标记的单链，再用于下游目标区域富集操作。
第三引物结合和线性扩增，即以捕获的单链为模板，使用第三引物进行单引物线性扩增，其中第三引物包括5’端的第三引物固定序列和3’端的目标区域特异性结合序列。
该步骤中，第三引物固定序列，是指这一段序列是固定不变的，而其它部分序列可以变化，因此“固定序列”的概念也可以称作“不变序列”或“通用序列”等。第三引物的3’端的目标区域特异性结合序列，能够特异性结合待富集目标区域，再通过PCR扩增实现目标区域的富集。
指数扩增，即以线性扩增产物为模板，使用第三引物和第一引物进行指数扩增，得到包含目标区域的产物。至此，实现了目标区域的富集。
目标区域的富集后产物可以应用于后续高通量测序或其它分析平台。本发明的方法，也同样可以应用于其它各种组织样本，甚至石蜡包埋甲醛固定的医学样本。
下面通过实施例详细说明本发明。
本实施例中所用的接头和引物序列如表1所示。
表1接头和引物序列
Figure PCTCN2015096790-appb-000001
Figure PCTCN2015096790-appb-000002
Figure PCTCN2015096790-appb-000003
注：①第一接头序列中下划线部分为用于标记不同样本的第一标签序列，“N”部分为用于标记不同目标区域分子序列的第二标签序列，其它部分为用于后续PCR扩增的第一接头固定序列(除与第一标签序列相邻的三个碱基以外)；②第三引物序列中下划线部分为3’端的目标区域特异性结合序列，其它部分为5’端的第三引物固定序列；③所有序列均按5’至3’形式列出。
本实施例具体实验过程如下：
1、将外周血肿瘤细胞样本DNA(100ng)进行末端修复后通过平末端或者粘末端方式连接特殊序列的接头(第一接头和第二接头)，并进行5-7个循环的扩增。其中，PCR扩增的一条引物(第一引物)上带有生物素修饰或其它可以进行特异链分离的标记。
1.1末端修复反应体系如下表2所示，20℃反应30min。
表2
组分 用量(μL)
片段化DNA X(100ng)
T4DNA聚合酶(NEB公司) 5
T4PNK(NEB公司) 5
dNTP混合物 1.6
T4PNK buffer(NEB公司)(10×) 10
总量 100
1.2连接反应体系如下表3所示，20℃反应30min。
表3
组分 用量(μL)
末端修复后DNA X
T4DNA连接酶(NEB公司) 1
T4DNA连接酶缓冲液(NEB公司) 5
第一接头 5
第二接头 5
ddH2O 余量
总量 50
1.3PCR扩增体系如下表4所示：
表4
组分 用量(μL)
DNA连接产物 X
dNTP(25mM) 3
Platinum pfx DNA polymerase(life technology) 0.6
第一引物 3
第二引物 3
MgSO4 4
10×pfx buffer 10
ddH2O 余量
总量 100
PCR扩增程序如下表5所示：
表5
Figure PCTCN2015096790-appb-000004
2、取上述连接有特殊接头(第一接头和第二接头)的PCR扩增产物1pmol，用链霉亲和素磁珠进行单链DNA的捕获，单链的捕获过程采用0.1M NaOH洗脱的方式。
3、针对上述捕获的生物素标记的PCR扩增产物单链，采用靶向结合目标区域的单引物(第三引物)进行线性扩增。线性扩增体系和程序分别如下表6和表7所示：
表6
组分 用量(μL)
捕获的单链DNA 6
5×Ion AmpliSeqTMHiFi Mix(life technology) 4
第三引物 10
总量 20
表7
Figure PCTCN2015096790-appb-000005
4、将线性扩增后的产物进行多重指数扩增，扩增引物的上游引物为1.3步骤的PCR扩增体系中生物素标记链特异匹配的引物(第一引物)，下游引物为线性扩增的引物组(第三引物)。多重指数扩增的体系和程序如下表8和表9所示：
表8
组分 用量(μL)
线性扩增后的DNA 6
5×Ion AmpliSeqTMHiFi Mix(life technology) 4
第一引物和第三引物 10
总量 20
表9
Figure PCTCN2015096790-appb-000006
5、最终的多重指数扩增后的产物纯化后进行上机测序分析，分析内容包括目标区域富集比例，覆盖均一性以及变异等内容。结果如下表10所示：
表10
参数 数值
样本 外周血肿瘤细胞样本
总的读取数目 1253091
靶数目 1009600
靶数目比例 80.56％
靶数目覆盖度 99.97％
目标区域捕获效率 66％
单碱基突变数目 43893
注：总的读取数目是指测序中读的到总的读段数目总数；靶数目指目标区域读取的读段数目总数；靶数目比例是指目标区域总数占总读取数目的比值；靶数目覆盖度指目标区域覆盖区域比例的情况；目标区域捕获效率指目标区域占整个参考基因组目标区域的比例情况；单碱基突变数目指目标区域检测到的 单个碱基突变数目的情况。
本申请还提供如下所示的另一实施例，以表明本方法的可行性。该实施例除了以肺癌样本代替外周血肿瘤细胞样本以外，其它方面包括实验过程和材料与上面的实施例相同。结果如下表11所示：
表11
参数 数值
样本 肺癌样本
总的读取数目 3163794
平均测序深度 32204.13
靶数目覆盖度 100.00％
目标区域捕获效率 89.10％
表12示出了在一个基因位置(chr7:55242469)上，采用常规杂交捕获建库和本发明的基于多重PCR的目标区域富集方法得到的等位基因频率的结果。
表12
Figure PCTCN2015096790-appb-000007
表11和表12的结果表明，本发明的基于多重PCR的目标区域富集方法在目标区域捕获效率上高达89.10％，能检出与常规杂交捕获建库同样的结果，且频率大致相近并且结果一致，由此可以确定本发明的方法可以应用于目标区域富集。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换。

Claims (10)

  1. 一种基于多重PCR的目标区域富集方法，包括如下步骤:
    在连接酶作用下，在含待富集目标区域的核酸片段两端分别连接第一接头和第二接头，得到接头连接产物，其中所述第一接头包括用于后续PCR扩增的第一接头固定序列、用于标记不同样本的第一标签序列和用于标记不同目标区域分子序列的第二标签序列；
    使用特异性结合所述第一接头固定序列的第一引物和特异性结合所述第二接头的第二引物，对所述接头连接产物进行PCR扩增，得到扩增产物，其中所述第一引物或第二引物具有第一亲和标记；
    通过固相载体捕获所述扩增产物中带有所述第一亲和标记的单链，其中所述固相载体上带有能够与所述第一亲和标记亲和结合的第二亲和标记；
    以捕获的所述单链为模板，使用第三引物进行单引物线性扩增，其中所述第三引物包括5’端的第三引物固定序列和3’端的目标区域特异性结合序列；
    以所述线性扩增产物为模板，使用所述第三引物和所述第一引物进行指数扩增，得到包含目标区域的产物。
  2. 根据权利要求1所述的基于多重PCR的目标区域富集方法，其特征在于，所述第一标签序列为5-10个碱基长度的序列，所述第二标签序列为10-12个碱基长度的随机序列。
  3. 根据权利要求1所述的基于多重PCR的目标区域富集方法，其特征在于，所述含待富集目标区域的核酸片段为末端修复过的核酸片段。
  4. 根据权利要求1所述的基于多重PCR的目标区域富集方法，其特征在于，所述第一引物具有第一亲和标记。
  5. 根据权利要求1所述的基于多重PCR的目标区域富集方法，其特征在于，所述第一亲和标记为生物素标记，所述第二亲和标记为链霉亲和素标记，所述固相载体为磁珠。
  6. 一种基于多重PCR的目标区域富集试剂，包括如下组成部分：
    第一接头和第二接头，用于在连接酶作用下分别连接到含待富集目标区域的核酸片段两端，以得到接头连接产物，其中所述第一接头包括用于后续PCR扩增的第一接头固定序列、用于标记不同样本的第一标签序列和用于标记不同目标区域分子序列的第二标签序列；
    第一引物和第二引物，用于对所述接头连接产物进行PCR扩增，得到扩增 产物，其中所述第一引物特异性结合所述第一接头固定序列，所述第二引物特异性结合所述第二接头，并且所述第一引物或第二引物具有第一亲和标记；
    固相载体，用于捕获所述扩增产物中带有所述第一亲和标记的单链，其中所述固相载体上带有能够与所述第一亲和标记亲和结合的第二亲和标记；
    第三引物，用于以捕获的所述单链为模板，进行单引物线性扩增，其中所述第三引物包括5’端的第三引物固定序列和3’端的目标区域特异性结合序列；
    其中，所述第三引物和所述第一引物还用于以所述线性扩增产物为模板，进行指数扩增，得到包含目标区域的产物。
  7. 根据权利要求6所述的基于多重PCR的目标区域富集试剂，其特征在于，所述第一标签序列为5-10个碱基长度的序列，所述第二标签序列为10-12个碱基长度的随机序列。
  8. 根据权利要求6所述的基于多重PCR的目标区域富集试剂，其特征在于，所述含待富集目标区域的核酸片段为末端修复过的核酸片段。
  9. 根据权利要求6所述的基于多重PCR的目标区域富集试剂，其特征在于，所述第一引物具有第一亲和标记。
  10. 根据权利要求6所述的基于多重PCR的目标区域富集试剂，其特征在于，所述第一亲和标记为生物素标记，所述第二亲和标记为链霉亲和素标记，所述固相载体为磁珠。
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