WO2021051378A1 - 测序文库的构建方法、测序方法及试剂盒和应用 - Google Patents

测序文库的构建方法、测序方法及试剂盒和应用 Download PDF

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WO2021051378A1
WO2021051378A1 PCT/CN2019/106928 CN2019106928W WO2021051378A1 WO 2021051378 A1 WO2021051378 A1 WO 2021051378A1 CN 2019106928 W CN2019106928 W CN 2019106928W WO 2021051378 A1 WO2021051378 A1 WO 2021051378A1
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stranded dna
sequencing
double
dna molecule
circular
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PCT/CN2019/106928
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French (fr)
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周荣芳
黄标
刘晨
周晏秋
黄金
田志坚
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武汉华大医学检验所有限公司
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Priority to CN201980087605.XA priority Critical patent/CN113646471A/zh
Priority to PCT/CN2019/106928 priority patent/WO2021051378A1/zh
Publication of WO2021051378A1 publication Critical patent/WO2021051378A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to the field of gene detection, in particular to a method for constructing a sequencing library, a sequencing method, a kit and an application.
  • the third-generation sequencing technology refers to single-molecule sequencing technology. When sequencing, PCR amplification is not required, and each DNA molecule is individually sequenced. It is mainly used for genome sequencing, methylation research, and mutation identification (SNP detection). )etc.
  • nanopore sequencing technology has the advantages of low cost, high throughput, and non-labeling.
  • the representative company is Oxford Nanopore Corporation.
  • the novel nanopore sequencing method uses electrophoresis technology to drive individual molecules through the nanopore one by one to achieve sequencing. Because the diameter of the nanopore is very small, only a single nucleic acid polymer is allowed to pass, and the charging properties of a single base of ATCG are different, and the type of the passed base can be detected by the difference in electrical signals, thereby realizing sequencing.
  • each base has many modifications such as methylation, so it also greatly increases the number of single bases. Base error rate.
  • each DNA molecule can only pass through the protein pore once and it will fail, so there is no way to shorten the length of the DNA molecule and perform base recognition in the form of CCS like the Pacbio platform.
  • an object of the present invention is to provide a method for constructing a sequencing library, a sequencing method, a kit and an application.
  • the sequencing library obtained by applying the construction method of the present invention is suitable for sequencing on a nanopore sequencing platform, can significantly improve the quality of sequencing, and reduce the error rate of a single base.
  • the motor protein When using the nanopore sequencing platform for sequencing, the motor protein is usually added to the DNA molecule along with the guide adapter. During the sequencing process, the motor protein will melt the double-stranded DNA, so that the single-stranded DNA will pass through the nanopore at a certain speed. . The sense strand and the antisense strand are completely separated, and they are sequenced separately during sequencing. In this way, for each double-stranded library, only one strand will be sequenced through the sequencing hole, and each base of the library can only be read once, and the single-base error rate of the nanopore sequencing platform is relatively high, which will lead to data failure. The reliability is low.
  • the inventors discovered during the research process that for linear double-stranded DNA samples, in the process of constructing a sequencing library, homologous recombination can be used to convert them into circular double-stranded DNA molecules, and then through the analysis of these circular double-stranded DNA samples.
  • the double-stranded DNA molecule is denatured and melted to obtain a circular single-stranded DNA molecule, which is used as a template for rolling circle amplification to obtain a sequencing library.
  • the formed sequencing library contains multiple consecutive linear sequences of the original circular single-stranded DNA molecule Copy of. Therefore, when using the nanopore sequencing platform for sequencing, the quality of sequencing can be significantly improved by sequencing these multiple consecutive linear copies.
  • the present invention provides a method for constructing a sequencing library, including: performing damage repair and end repair on a linear double-stranded DNA sample to obtain a repaired DNA sample; The sample undergoes homologous recombination to obtain a circular double-stranded DNA molecule; the circular single-stranded DNA molecule denatured by the circular double-stranded DNA molecule is used as a template to perform rolling circle amplification to obtain an RCA amplification product; The RCA amplification product is connected with a sequencing adapter to obtain the sequencing library.
  • the present invention connects the "linear double-stranded DNA” into a "circular double-stranded DNA molecule" by homologous recombination, and then uses the denatured circular single-stranded DNA molecule of the circular double-stranded DNA molecule as a template, and uses rolling Change the amplification method to form multiple consecutive linear copies of the template molecule, and then proceed to library construction and sequencing, thereby improving the quality of sequencing.
  • the method for constructing a sequencing library described above may further include the following technical features:
  • homologous recombinase is used to perform the homologous recombination.
  • it further comprises: before homologous recombination of the repaired DNA sample, connecting oligonucleotide sequences at both ends of the repaired DNA sample.
  • an identical oligonucleotide sequence can be connected to both ends of the repaired DNA sample to facilitate subsequent homologous recombination.
  • the length and arrangement requirements of the oligonucleotide sequence connected at both ends can be determined according to the homologous recombinase used, and different homologous recombinases can recognize different oligonucleotide sequences. For example, taking NEBuilder homologous and same set of enzymes as an example, a chain of the oligonucleotide sequence recognized by it is shown in SEQ ID NO:1. If other homologous recombinases with similar functions are used, the corresponding oligonucleotide sequence needs to be modified.
  • oligonucleotide sequence it is also possible to connect a different oligonucleotide sequence to the two ends of the repaired DNA sample. Since the oligonucleotide sequence connected to the two ends of the DNA sample is different, it is also possible to perform homologous recombination. It is necessary to introduce another oligonucleotide sequence, which can be complementary to the 5'end and 3'end sequence of the DNA sample connected with the oligonucleotide sequence.
  • the repaired DNA sample is purified before the repaired DNA sample is subjected to homologous recombination.
  • the circular double-stranded DNA molecule is purified before rolling circle amplification is performed using the circular single-stranded DNA molecule denatured by the circular double-stranded DNA molecule.
  • the amplification product is purified before the amplification product is connected to the sequencing adapter.
  • the obtained connection product is purified.
  • magnetic beads are used for the purification.
  • the use of magnetic beads for purification can effectively remove unnecessary impurities and further improve the quality of sequencing.
  • high-temperature denaturation can be used to denature circular double-stranded DNA molecules into circular single-stranded DNA molecules, which facilitates subsequent primer binding and RCA reaction efficiency.
  • it can be denatured at 95 degrees Celsius for 2 to 5 minutes to achieve denaturation of circular double-stranded DNA molecules into circular single-stranded DNA molecules.
  • BST DNA polymerase is used to perform the rolling circle amplification.
  • the denaturation process of the circular double-stranded DNA molecule can be denatured into the circular single-stranded DNA molecule, and the rolling circle amplification process using the circular single-stranded DNA molecule as a template.
  • the temperature of the denaturation process is generally 90 to 95 degrees Celsius, this requires the DNA polymerase used in the rolling circle amplification process to have thermal stability.
  • BST DNA polymerase is derived from Bacillus stearothermophilus.
  • the BST DNA polymerase can be directly purchased commercially, wild-type BST DNA polymerase can be purchased directly, or BST 2.0 DNA polymerase or BST 3.0 DNA polymerase (ie homologs of BST DNA polymerase) can be purchased, some Compared with the wild-type Bst DNA polymerase, the commercially available BST 2.0 DNA polymerase or BST 3.0 DNA polymerase has improved amplification rate, yield, salt tolerance, and thermal stability.
  • BST DNA polymerase can also be prepared by itself.
  • the structural gene of Bst DNA polymerase large fragment can be cloned and isolated from a high-temperature-resistant Bacillus stearothermophilus specific strain, and then recombined into an expression plasmid vector.
  • Some prokaryotic expression systems, such as E. coli, perform stable and high-efficiency expression to obtain the corresponding BST DNA polymerase.
  • Rolling circle amplification using BST DNA polymerase can quickly obtain a large number of circular amplification products for sequencing on the nanopore platform.
  • the sequencing library is suitable for nanopore platform sequencing.
  • the present invention provides a sequencing method.
  • the sequencing method includes: obtaining a sequencing library based on the construction method described in any one of the embodiments of the first aspect of the present invention, and using nanopore sequencing technology to The sequencing library is sequenced.
  • the present invention provides a kit comprising: a homologous recombinase, a BST DNA polymerase, and an oligonucleotide sequence.
  • the kit further includes at least one of the following: end repair and/or damage repair, magnetic beads, sequencing adapters, RCA primers, the RCA primers such as SEQ ID NO: 2 shown.
  • the oligonucleotide sequence is a double-stranded DNA molecule, one strand of the oligonucleotide sequence is shown in SEQ ID NO:1, and the other strand of the oligonucleotide sequence It is the complementary sequence of SEQ ID NO:1.
  • the present invention provides a method for preparing a circular double-stranded DNA molecule, comprising: providing a linear double-stranded DNA molecule and an oligonucleotide sequence; linking the oligonucleotide sequence to the The two ends of the linear double-stranded DNA molecule obtain a first ligation product; the first ligation product generates a homologous recombination splicing product under the action of a homologous recombinase, that is, the circular double-stranded DNA is obtained.
  • the circular double-stranded DNA molecule thus obtained can be subsequently applied to the field of second-generation sequencing or third-generation sequencing.
  • the homologous recombinase is NEBuilder homologous recombinase
  • the oligonucleotide sequence is a double-stranded DNA molecule
  • one strand of the oligonucleotide sequence is as SEQ ID NO:
  • the other strand of the oligonucleotide sequence is the complementary sequence of SEQ ID NO:1.
  • the present invention provides the application of homologous recombination in RCA amplification.
  • the application includes: linear double-stranded DNA molecules are prepared by homologous recombination to obtain circular double-stranded DNA molecules; A circular single-stranded DNA molecule denatured by a double-stranded DNA molecule is used as a template for rolling circle amplification.
  • the present invention provides a method for constructing a sequencing library, which includes: performing damage repair and end repair on a linear double-stranded DNA sample to obtain a repaired DNA sample; and performing homologous recombination on the repaired DNA sample to obtain A circular double-stranded DNA molecule; using the denatured circular single-stranded DNA molecule of the circular double-stranded DNA molecule as a template to perform rolling circle amplification to obtain an amplified product; connect the amplified product to a sequencing adapter, In order to obtain the sequencing library.
  • homologous recombination generally refers to anything that occurs between gene sequences, or occurs in a piece of gene sequence itself, by means of the interaction of at least part of the homologous sequences on the gene sequence, so that the gene sequence is spliced. process.
  • DNA ligase can be used to ligate an oligonucleotide sequence at both ends of the repaired DNA sample to obtain a ligation product. Then homologous recombination is carried out by means of homologous recombinase to obtain circular DNA molecules.
  • the homologous recombinase of NEBuilder can be used to mix the ligation product and the homologous recombinase, and react overnight at 37 degrees Celsius to obtain the circular double-stranded DNA molecule.
  • NEB builder’s homologous recombinase has exonuclease activity that enables the ends of linear double-stranded molecules formed by linear double-stranded DNA samples to continue to be digested during the process of homologous recombination, thereby avoiding linear double-stranded DNA samples.
  • the circular double-stranded DNA molecule formed by homologous recombination will not be affected by the exonuclease activity of the homologous recombinase.
  • the circularization efficiency of linear double-stranded DNA samples can be greatly improved. Under the reaction conditions, self-ligation products are formed, that is, circular double-stranded DNA molecules with high yield can be obtained.
  • the obtained circular DNA molecule is used as a template, and DNA polymerase and primers are used for amplification.
  • rolling circle amplification uses single-stranded circular DNA molecules as templates, circular double-stranded DNA molecules can be amplified while melting.
  • the denaturing solution, DNA polymerase and RCA primers can be added in the same reaction system, so that the circular double-stranded DNA molecule can be melted into single-stranded, and RCA amplification can be realized.
  • BST DNA polymerase can be used to perform the rolling circle amplification.
  • the reaction system can be incubated at 65 degrees Celsius for 30 to 60 minutes to obtain a large amount of amplified products.
  • the present invention provides a kit, which can be used for the construction and sequencing of a sample library.
  • the kit includes: homologous recombinase, BST DNA polymerase, and oligonuclease Nucleotide sequence.
  • the oligonucleotide sequence and homologous recombinase in the kit can convert linear double-stranded DNA samples into circular double-stranded DNA molecules.
  • the circular double-stranded DNA molecule is denatured into a single-stranded circular DNA template, and rolling circle amplification is performed with the help of BST DNA polymerase and primers to obtain a large number of amplified products, which are applied to the sequencing library.
  • the kit may further include at least one of the following: end repair and/or damage repair, magnetic beads, and sequencing adapters.
  • the oligonucleotide sequence used is a double-stranded DNA molecule, in which the sequence of one strand is shown in SEQ ID NO:1, and the other strand is the complementary sequence of SEQ ID NO:1.
  • the sequencing adaptor used can be any adaptor that can be used in the nanopore sequencing platform, for example, it can be the LSK109 sequencing adaptor.
  • the first method is based on lambda DNA as the starting sample. After damage repair and end repair, homologous recombination is performed to obtain a circular double-stranded DNA molecule. Then, after connecting the ONT 1D linker, the ONT platform sequencing is performed, and the next Machine data for analysis. Specific steps are as follows:
  • the reagents used are from commercially available kits:
  • NEB M6630L NEB Next FFPE DNA Repair Mix 96 reaction NEB (purchased from NEB, catalog number: 1000002572);
  • step (1) Refer to the following steps to purify the repaired product obtained in step (1) to obtain a purified product.
  • thermomixer 400 rpm or vertical mixer
  • the magnetic beads used were purchased from Agencourt AMPure XP Beads, AGENCOURT, and the article number is A63881.
  • Reagent volume DNA purified in the previous step (about 600ng) 22.5 ⁇ L Oligonucleotide sequence 2.5 ⁇ L Blunt/TA Ligase Master Mix 25 ⁇ L Total 50 ⁇ L
  • the Blunt/TA Ligase Master Mix reagent was purchased from the commercial kit NEB Blunt/TA Ligase Master Mix, the item number is M0367L.
  • the oligonucleotide sequence used was synthesized by Shanghai Shenggong Biotechnology Co., Ltd.
  • the oligonucleotide sequence is a double-stranded DNA molecule, one of the strands is shown in SEQ ID NO:1, and the other strand is its complementary strand.
  • the oligonucleotide sequence is as follows:
  • the NEBuilder HiFi Master Mix reagent used comes from a commercially available kit, which is High-fidelity DNA assembly premix (manufacturer is NEB, item number is 1000009200).
  • the obtained double-stranded circular DNA molecules were purified with 0.6 times magnetic beads (purchased from Novozan VAHTS DNA Clean Beads, catalog number N411), and after detecting the concentration, the following rolling circle amplification was performed.
  • step (4) Using the homologous recombination splicing product obtained in step (4), configure the following reaction system, denature at 95°C for 2 min, and then incubate at 65°C for 30-60 min (adjust the reaction time slightly according to the fragment size) to obtain the amplified product.
  • the 10 ⁇ Isothermal Amplification Buffer II used is also from the kit High-fidelity DNA assembly premix (manufacturer is NEB, item number is 1000009200).
  • the RCA primer sequence used is: 5'-NNNGCAATACGTAACTGAACGAAGTNNN-3' (SEQ ID NO: 2), synthesized by Shanghai Shenggong Biotechnology Co., Ltd.
  • N in SEQ ID NO: 2 represents any base A, T, C, or G.
  • three N bases can be added to both ends of the designed RCA primers. In this way, the binding efficiency of RCA primers and single-stranded circular DNA molecules can be improved.
  • the reagent Adapter Mix comes from the commercial kit SQK-LSK109# connection sequencing kit (ONT official reagent, catalog number: 1000002572).
  • the reagents Ligation Buffer and NEBNext Quick T4 DNA Ligase come from the commercial kit E6056L NEBNext Quick T4 DNA Ligase 100 reaction NEB (NEB, catalog number: 1000007268).
  • the ligation product was purified using a kit purchased from Agencourt AMPure XP Beads of AGENCOURT company with the article number A63881:
  • the average insert length refers to the average length of all the sequenced sequences after removing the linker.
  • the experimentally measured insert length is greater than 15kb to be qualified, that is, only when the measured average insert length is greater than 15kb, can it be Complete advanced information analysis such as gene assembly.
  • the Q value is a measure of the sequencing process by the ONT sequencer machine after the sequencing is completed.
  • the experimental determination that the Q value is greater than 7 is qualified (refer to the official definition of ONT).
  • the Q value is less than 7, it generally indicates that there is a problem in the sequencing process. , The accuracy of a single base is less than 90%, and sequencing data is often unavailable; and between 1-12, the higher the Q value, the better the quality.
  • This second method refers to the official ONT experimental method.
  • the starting sample is lambda DNA.
  • the ONT 1D adapter is connected, and the ONT platform sequencing is performed, and the offline data is analyzed.
  • kits used in this method can refer to the kits in the first method, which are not specifically listed and explained.
  • thermomixer 400 rpm or vertical mixer
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.

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Abstract

提供了一种测序文库的构建方法、测序方法及试剂盒和应用。该测序文库的构建方法包括将线性双链DNA样本进行损伤修复和末端修复，以便获得经修复的DNA样本；将该经修复的DNA样本进行同源重组，以便获得环状双链DNA分子；以环状双链DNA分子变性的环状单链DNA分子为模板，进行滚环扩增，以便获得RCA扩增产物；RCA扩增产物与测序接头连接，以便获得该测序文库。还提供了测序方法、试剂盒、制备环状双链DNA分子的方法以及同源重组在RCA扩增中的应用。

Description

测序文库的构建方法、测序方法及试剂盒和应用 技术领域
本发明涉及基因检测领域，具体涉及一种测序文库的构建方法、测序方法及试剂盒和应用。
背景技术
第三代测序技术是指单分子测序技术，在测序时，不需要经过PCR扩增，实现了对每一条DNA分子的单独测序，主要应用于基因组测序、甲基化研究、突变鉴定(SNP检测)等方面。
纳米孔测序技术作为第三代测序技术中的一项重要的技术，具有成本低、高通量以及非标记等优势。其中代表性的公司为英国牛津纳米孔公司。新型纳米孔测序法(nanopore sequencing)是采用电泳技术，借助电泳驱动单个分子逐一通过纳米孔来实现测序的。由于纳米孔的直径非常细小，仅允许单个核酸聚合物通过，而ATCG单个碱基的带电性质不一样，通过电信号的差异就能检测出通过的碱基类别，从而实现测序。
在测序的过程中，DNA通过蛋白测序孔速度极快(450bp/s)，加之DNA上面除了ATGC四种碱基，每种碱基还有甲基化等诸多的修饰，所以也大大增加了单个碱基的错误率。而且每个DNA分子只能通过一次蛋白孔就会失效，所以也没有办法像Pacbio平台一样，通过缩短DNA分子长度，以CCS形式进行碱基识别。
因此，基于ONT的纳米孔测序技术的发展受到了极大的限制。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出一种测序文库的构建方法、测序方法及试剂盒和应用。应用本发明的构建方法所获得的测序文库，适合于纳米孔测序平台进行测序，能够显著提高测序质量，降低单个碱基的错误率。
在利用纳米孔测序平台进行测序时，通常会将马达蛋白随引导接头一同加在DNA分子上，在测序过程中，马达蛋白会对双链DNA解链，使得单链DNA以一定速度经过纳米孔。正义链与反义链是完全分离开的，在测序的时候是分别被单独测序的。这样针对每个双链的文库，只有一条链会通过测序孔进行测序，所有该文库每个碱基只能读取一次，且纳米孔测序平台单碱基错误率较高，从而会导致数据可信度较低。
为此，发明人在研究过程中发现，针对线性双链DNA样本，在构建测序文库的过程中， 可以利用同源重组的方式将其转换成环状双链DNA分子，然后通过对这些环状双链DNA分子变性解链，获得环状单链DNA分子，并以此为模板进行滚环扩增，获得测序文库，所形成的测序文库中包含原始环状单链DNA分子的多个连续线性的拷贝。由此在利用纳米孔测序平台测序时，通过对这些多个连续线性的拷贝进行测序，显著提高测序的质量。
具体而言，本申请提供了如下技术方案：
在本发明的第一方面，本发明提供了一种测序文库的构建方法，包括：将线性双链DNA样本进行损伤修复和末端修复，以便获得经修复的DNA样本；将所述经修复的DNA样本进行同源重组，以便获得环状双链DNA分子；以所述环状双链DNA分子变性的环状单链DNA分子为模板，进行滚环扩增，以便获得RCA扩增产物；将所述RCA扩增产物与测序接头连接，以便获得所述测序文库。本发明通过将“线性双链DNA”通过同源重组连接的方式连接成“环状双链DNA分子”，然后以该环状双链DNA分子变性的环状单链DNA分子为模板，采用滚换扩增的方式，形成模板分子的多个连续的线性拷贝，然后进行建库和测序，从而提高测序质量。
根据本发明的实施例，以上所述测序文库的构建方法可以进一步包括如下技术特征：
在本发明的一些实施例中，利用同源重组酶进行所述同源重组。
在本发明的一些实施例中，进一步包括：在将所述经修复的DNA样本进行同源重组之前，在所述经修复的DNA样本的两端连接寡核苷酸序列。
在同源重组之前，可以在经修复的DNA样本的两端连接上一段相同的寡核苷酸序列，方便后续的同源重组。两端所连接的寡核苷酸序列长度和排列要求可以依据所用到的同源重组酶而确定，不同的同源重组酶可以识别不同的寡核酸序列。例如以NEBuilder同源同组酶为例，其所识别的寡核苷酸序列的一条链如SEQ ID NO:1所示。如果采用如果使用其他功能类似的同源重组酶，则需要修改对应的寡核苷酸序列。当然，也快可以在经修复的DNA样本的两端分别连接上一段不同的寡核苷酸序列，由于DNA样本的两端所连接的寡核苷酸序列不同，在进行同源重组时，还需要引入另一寡核苷酸序列，该寡核苷酸序列能够和连接有寡核苷酸序列的DNA样本的5’端和3’端序列互补配对。
在本发明的一些实施例中，在获得经修复的DNA样本之后，在将所述经修复的DNA样本进行同源重组之前，对所述经修复的DNA样本进行纯化。
在本发明的一些实施例中，在以所述环状双链DNA分子变性的环状单链DNA分子为模板，进行滚环扩增之前，对所述环状双链DNA分子进行纯化。
在本发明的一些实施例中，在获得所述扩增产物之后，在将所述扩增产物与测序接头连接之前，对所述扩增产物进行纯化。
在本发明的一些实施例中，在将所述扩增产物与所述测序接头连接之后，对获得的连 接产物进行纯化。
在本发明的一些实施例中，利用磁珠进行所述纯化。采用磁珠进行纯化，能够有效去除不必要的杂质，进一步提高测序的质量。
在本发明的一些实施例中，利用高温变性，能够将环状双链DNA分子变性成环状单链DNA分子，便于后续引物结合和RCA反应效率。例如可以在95摄氏度条件下变性2～5分钟，实现将环状双链DNA分子变性为环状单链DNA分子。
在本发明的一些实施例中，利用BST DNA聚合酶进行所述滚环扩增。在操作过程中，为了提高效率，方便操作，可以将环状双链DNA分子变性成环状单链DNA分子的变性过程，以及以环状单链DNA分子为模板进行滚环扩增的过程，在同一个反应体系中进行。考虑到变性过程的温度一般在90～95摄氏度，这就要求在滚环扩增过程中所用到的DNA聚合酶具有热稳定性。BST DNA聚合酶来源于嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)，是一种具有5'→3'的聚合酶活性和双链特异的5'→3'外切酶活性，但没有3'→5'外切酶活性的大片段，其具有高扩增效率和热稳定性。该BST DNA聚合酶可以直接通过商购获得，可以直接购买野生型的BST DNA聚合酶，也可以购买BST 2.0DNA聚合酶或者BST 3.0DNA聚合酶(即BST DNA聚合酶的同源物)，一些市售的BST 2.0DNA聚合酶或者BST3.0DNA聚合酶和野生型Bst DNA聚合酶相比，具有提高的扩增速率、产量、耐盐性和热稳定性等。当然BST DNA聚合酶也可以自己制备获得，例如可以从耐高温的嗜热脂肪芽孢杆菌特异菌株中克隆分离获得Bst DNA聚合酶大片段的结构基因，然后将其重组于表达质粒载体上，然后在一些原核表达系统，例如大肠杆菌中进行稳定的高效表达，获得相应的BST DNA聚合酶。利用BST DNA聚合酶进行滚环扩增，可以快速获得大量环状扩增产物，用于纳米孔平台测序。
在本发明的一些实施例中，所述测序文库适合于纳米孔平台测序。
在本发明的第二方面，本发明提供了一种测序方法，所述测序方法包括：基于本发明第一方面任一实施例所述的构建方法，获得测序文库，利用纳米孔测序技术对所述测序文库进行测序。
在本发明的第三方面，本发明提供了一种试剂盒，所述试剂盒包括：同源重组酶，BST DNA聚合酶，和寡核苷酸序列。
在本发明的一些实施例中，所述试剂盒进一步包括下列中的至少一种：末端修复物和/或损伤修复物，磁珠，测序接头，RCA引物，所述RCA引物如SEQ ID NO:2所示。
在本发明的一些实施例中，所述寡核苷酸序列为双链DNA分子，所述寡核苷酸序列的一条链如SEQ ID NO:1所示，所述寡核酸序列的另一条链为SEQ ID NO:1的互补序列。
在本发明的第四方面，本发明提供了一种制备环状双链DNA分子的方法，包括：提供 线性双链DNA分子和寡核苷酸序列；将所述寡核苷酸序列连接到所述线性双链DNA分子的两端，获得第一连接产物；所述第一连接产物在同源重组酶的作用下生成同源重组拼接产物，即获得所述环状双链DNA。由此获得的环状双链DNA分子，后续可以应用于二代测序或者三代测序领域中。
在本发明的一些实施例中，所述同源重组酶为NEBuilder同源重组酶，所述寡核苷酸序列为双链DNA分子，所述寡核苷酸序列的一条链如SEQ ID NO:1所示，所述寡核酸序列的另一条链为SEQ ID NO:1的互补序列。
在本发明的第五方面，本发明提供了同源重组在RCA扩增中的应用，所述应用包括：线性双链DNA分子通过同源重组制备得到环状双链DNA分子；所述环状双链DNA分子变性的环状单链DNA分子为模板，进行滚环扩增。
具体实施方式
下面对本发明的实施例进行详细描述，所描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。对于本文中一些术语的解释，仅仅是方便本领域技术人员的理解，不应看做是对本发明的限制。
本发明提供了一种测序文库的构建方法，包括：将线性双链DNA样本进行损伤修复和末端修复，以便获得经修复的DNA样本；将所述经修复的DNA样本进行同源重组，以便获得环状双链DNA分子；以所述环状双链DNA分子变性的环状单链DNA分子为模板，进行滚环扩增，以便获得扩增产物；将所述扩增产物与测序接头连接，以便获得所述测序文库。
本文中，术语“同源重组”泛指任何发生在基因序列之间的，或者发生在一段基因序列自身的，借助于基因序列上的至少部分同源序列的相互作用，使得基因序列发生拼接的过程。
在本发明的至少一些实施方式中，可以利用DNA连接酶，在经修复的DNA样本的两端均连接一段寡核苷酸序列，获得连接产物。然后借助于同源重组酶进行同源重组，获得环状DNA分子。在本发明的至少一些实施方式中，可以借助于NEBuilder的同源重组酶，将连接产物和同源重组酶混合，在37摄氏度条件下反应过夜，获得该环状双链DNA分子。NEB公司builder的同源重组酶，在同源重组的过程中，其所具有的外切酶活性能够使得线性双链DNA样本形成的线性双链分子的末端会继续被消化，从而避免了线性双链分子的形成。而经过同源重组所形成的环状双链DNA分子不会受到同源重组酶的外切酶活性的影响。从而可以大大提高线性双链DNA样本的环化效率，在该反应条件下，形成自连产物，即获得高得率的环状双链DNA分子。
在进行滚环扩增时，以所获得的环状DNA分子为模板，利用DNA聚合酶和引物进行扩增。由于滚环扩增是以单链环状DNA分子为模板，所以针对环状双链DNA分子，可以边解链边进行扩增。例如，可以在同一个反应体系中，加入变性溶液、DNA聚合酶和RCA引物，从而可以实现环状双链DNA分子解链为单链，以及RCA扩增。在本发明的至少一些实施方式中，可以利用BST DNA聚合酶进行所述滚环扩增。借助于BST DNA聚合酶进行滚环扩增时，可以将反应体系在65摄氏度条件下孵育30～60分钟，获得大量扩增产物。
在本发明的另一方面，本发明提供了一种试剂盒，应用该试剂盒可以用于样本文库的构建和测序，所述试剂盒包括：同源重组酶，BST DNA聚合酶，和寡核苷酸序列。应用试剂盒中的寡核苷酸序列和同源重组酶可以将线性双链DNA样本转换为环状双链DNA分子。将环状双链DNA分子变性为单链环状DNA模板，借助于BST DNA聚合酶和引物，进行滚环扩增，获得大量扩增产物，应用于测序文库。另外，所述试剂盒还可以进一步包括下列中的至少一种：末端修复物和/或损伤修复物，磁珠，测序接头。在至少一些实施方式中，所用到的寡核苷酸序列为双链DNA分子，其中的一条链的序列如SEQ ID NO:1所示，另一条链为SEQ ID NO:1的互补序列。所用到的测序接头可以是能够用于纳米孔测序平台的任意接头，例如可以是LSK109测序接头。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
基于lambda DNA，分别采用两种不同的方法构建测序文库，然后进行ONT平台测序，并获得相应的数据。
一、第一种方法
该第一种方法基于lambda DNA作为起始样品，经损伤修复和末端修复后，进行同源重组，获得环状双链DNA分子，然后连接上ONT 1D接头后，进行ONT平台测序，并对下机数据进行分析。具体步骤如下：
(1)DNA的损伤修复和末端修复：
取1.2μg原始核酸，参照下表，在PCR仪中孵育，进行损伤修复和末端修复，程序如下：20℃ 7min；65℃ 7min，获得修复后的产物。
试剂 体积
1.2μg DNA in Nuclease-Free water 48μL
NEBNext FFPE DNA Repair Buffer 3.5μL
NEBNext FFPE DNA Repair Mix 2μL
Ultra II End-prep reaction buffer 3.5μL
Ultra II End-prep enzyme mix 3μL
Total 60μL
其中所用到的各试剂来自于商购试剂盒：
M6630L NEBNext FFPE DNA Repair Mix 96 reaction NEB(购自于NEB，货号：1000002572)；
E7546L Ultra II End-prep enzyme mix 96 reaction NEB(购自于NEB，货号：1000003887)。
(2)修复后纯化
参照下述步骤，将步骤(1)所获得的修复后的产物进行纯化，获得纯化后产物。
a)取60μL重悬均匀后的AMPure XP beads于1.5mL低吸附离心管中；
b)将孵育后的DNA样本(即上述修复后的产物)60μL全部转移至已加入磁珠的1.5ml低吸附离心管中，轻弹混匀；
c)将离心管放置于thermomixer 400rpm(或垂直混匀仪)上室温孵育5min；
d)使用新鲜Nuclease-free water配制500μL 70体积％的乙醇；
e)待混匀结束后将离心管取下，瞬时离心后放置于磁力架上，液体澄清后去掉上清(避免洗到磁珠)；
f)保持离心管在磁力架上，从磁珠上方缓慢加入200μL配好的70％乙醇，待乙醇全部流至管底后吸出全部乙醇；
g)重复步骤f)；
h)取下离心管，瞬时离心后重新放置于磁力架，换用10μL枪头吸走残留液体，室温干燥30秒，注意不要让磁珠干裂；
i)取下离心管，加入15μL新鲜NF-Water重悬磁珠，室温孵育2min。
所用到的磁珠购自于AGENCOURT公司Agencourt AMPure XP Beads，货号为A63881。
(3)连接寡核苷酸序列
按照下表的顺序添加试剂，每个试剂添加完后要轻弹离心管混匀：
试剂 体积
上一步纯化后的DNA(约600ng) 22.5μL
寡核苷酸序列 2.5μL
Blunt/TA Ligase Master Mix 25μL
Total 50μL
轻弹离心管以混匀各组分，然后瞬时离心，在thermomixer上室温孵育60min，获得连 接有寡核苷酸序列的产物。其中Blunt/TA Ligase Master Mix试剂购自于商用试剂盒NEB Blunt/TA Ligase Master Mix，货号为M0367L。
其中所用到的寡核苷酸序列，由上海生工生物技术有限公司合成。该寡核苷酸序列为双链DNA分子，其中一条链序列为SEQ ID NO:1所示，另一条链为其互补链，该寡核苷序列如下：
5'-GCAATACGTAACTGAACGAAGT-3'(SEQ ID NO:1)
3'-CGTTATGCATTGACTTGCTTCA-5'。
(4)同源重组
取上述步骤(3)所获得的连接有寡核苷酸序列的产物，重新进行qubit定量，然后至少取样600ng(如果根据qubit的取样体积大于20μl，则需要进行磁珠纯化浓缩体积，然后进行同源重组反应)，采用NEBuilder同源重组酶，37℃过夜反应，反应体系如下表，获得同源重组拼接产物。
反应组份 体积(μL)
连接有寡核苷酸序列的产物 X
NEBuilder HiFi Master Mix(含有NEBuilder同源重组酶) 20
水 20-X
总体积 40
其中所用到的NEBuilder HiFi Master Mix试剂来自于商购试剂盒，该试剂盒为
Figure PCTCN2019106928-appb-000001
高保真DNA组装预混液(厂家为NEB，货号为1000009200)。
对于所获得的双链环状DNA分子用0.6倍磁珠(购自于诺唯赞VAHTSTM DNA Clean Beads，货号为N411)进行纯化，检测浓度后，进行下述滚环扩增。
(5)RCA扩增
利用步骤(4)所获得的同源重组拼接产物，配置如下反应体系，95℃变性2min，然后65℃孵育30-60min(依据片段大小略微调整反应时间)，获得扩增产物。
试剂 体积(μL)
同源重组拼接产物 40
10×Isothermal Amplification Buffer II 5
MgSO 4 1
RCA引物 1
dNTP(10μM) 2
Bst 3.0 1
其中所用到10×Isothermal Amplification Buffer II也来自于试剂盒
Figure PCTCN2019106928-appb-000002
高保真DNA组装预混液(厂家为NEB，货号为1000009200)。
反应完成后，使用1X体积磁珠纯化RCA产物，用61μL水回融。
其中所用到的RCA引物序列为：5'-NNNGCAATACGTAACTGAACGAAGTNNN-3'(SEQ ID NO:2)，由上海生工生物技术有限公司合成。
其中SEQ ID NO:2中N代表任意碱基A、T、C或者G。在设计RCA引物时，所设计的RCA引物的两端可以各添加三个N碱基，通过这种方式可以提高RCA引物与单链环状DNA分子的结合效率。
(6)建库：加接头LSK109
参照下表配置反应体系后，轻弹离心管以混匀各组分，瞬时离心，室温孵育30min，获得连接产物。
试剂 体积
上述步骤(5)的扩增产物 60μL
Ligation Buffer(LNB) 25μL
NEBNext Quick T4 DNA Ligase 10μL
Adapter Mix(AMX) 5μL
Total 100μL
其中，试剂Adapter Mix来自于商用试剂盒SQK-LSK109#连接测序试剂盒(ONT官方试剂，货号：1000002572)。
试剂Ligation Buffer和NEBNext Quick T4 DNA Ligase来自于商用试剂盒E6056L NEBNext Quick T4 DNA Ligase 100 reaction NEB(NEB,货号：1000007268)。
然后采用购自于AGENCOURT公司Agencourt AMPure XP Beads，货号为A63881的试剂盒，对所述连接产物进行纯化：
a)待孵育完成后，加入40μL重悬AMPure XP beads并轻弹离心管混匀；
b)将离心管放置于垂直混匀仪上室温孵育5min；
c)瞬时离心后放置于磁力架上，待液体澄清后去掉上清；
d)取下离心管，加入250μL Fragment Buffer(LFB)并轻弹至完全混匀，瞬时离心后重新放置于磁力架上，待液体澄清后去掉上清；
e)重复步骤d；
f)取下离心管，瞬时离心后重新放置于磁力架上，待液体澄清后用小体积的吸头吸出残留液体；
g)取下离心管，加入25μLElution Buffer(EB)轻弹混匀，室温孵育10min；
h)将离心管瞬时离心后，放置于磁力架上，待液体澄清后取出25μL上清于新的1.5mL 低吸附离心管中，取1μL测定浓度，计算得率。
(7)采用ONT Premethion平台，进行R 9.4芯片的测序，获得测序结果。
采用第一种方法所获得的测序结果如下：
下机数据量：66Gb(大于50G b即为合格)
平均插入片段长度：22Kb。
Q值：10.5。
其中平均插入片段长度是指去除了接头之后序列的所有测序序列长度的平均值，实验测得插入片段的长度大于15kb即为合格，即只有当测得的平均插入片段长度大于15kb的时候，才能完成基因组装等高级信息分析。
Q值是ONT测序仪机器在测序完成之后对测序过程的一种衡量方式，实验测定Q值大于7即为合格(参照ONT官方定义)，当Q值小于7的时候，一般指示测序过程出现问题，单个碱基的正确率小于90％，测序数据往往是不可用的；而且在1-12之间，Q值越高，表示质量越好。
第二种方法：
该第二种方法参照ONT官方实验方法，起始样品为lambda DNA，经损伤修复和末端修复后，连接上ONT 1D接头后，进行ONT平台测序，并对下机数据进行分析。该方法中所用到的一些商购试剂盒可参照第一种方法中的试剂盒，未专门列出和说明。
具体步骤如下：
(1)DNA的损伤修复和末端修复：
取1.2μg原始核酸，参照下表，在PCR仪中孵育，进行损伤修复和末端修复，程序如下：20℃ 7min；65℃ 7min，获得修复产物。
试剂 体积
1.2μg DNA in Nuclease-Free water 48μL
NEBNext FFPE DNA Repair Buffer 3.5μL
NEBNext FFPE DNA Repair Mix 2μL
Ultra II End-prep reaction buffer 3.5μL
Ultra II End-prep enzyme mix 3μL
Total 60μL
(2)修复后纯化
参照下述步骤，将步骤(1)获得的修复产物进行纯化，获得纯化产物：
a)取60μL重悬均匀后的AMPure XP beads于1.5mL低吸附离心管中；
b)将孵育后的DNA样本60μL全部转移至已加入磁珠的1.5ml低吸附离心管中，轻弹混匀；
c)将离心管放置于thermomixer 400rpm(或垂直混匀仪)上室温孵育5min；
d)使用新鲜Nuclease-free water配制500μL 70％乙醇；
e)待混匀结束后将离心管取下，瞬时离心后放置于磁力架上，液体澄清后去掉上清(避免洗到磁珠)；
f)保持离心管在磁力架上，从磁珠上方缓慢加入200μL配好的70％乙醇，待乙醇全部流至管底后吸出全部乙醇；
g)重复步骤f)；
h)取下离心管，瞬时离心后重新放置于磁力架，换用10ul枪头吸走残留液体，室温干燥30秒，注意不要让磁珠干裂；
i)取下离心管，加入15μL新鲜NF-Water重悬磁珠，室温孵育2min。
(3)建库：加接头LSK109
参照下表配置反应体系后，轻弹离心管以混匀各组分，瞬时离心，室温孵育5min，获得连接产物。
试剂 体积
步骤(2)获得的纯化产物 60μL
Ligation Buffer(LNB) 25μL
NEBNext Quick T4 DNA Ligase 10μL
Adapter Mix(AMX) 5μL
Total 100μL
然后参照下述步骤，对所获得的连接产物进行纯化：
a)待孵育完成后，加入40μL重悬AMPure XP beads并轻弹离心管混匀；
b)将离心管放置于垂直混匀仪上室温孵育5min；
c)瞬时离心后放置于磁力架上，待液体澄清后去掉上清；
d)取下离心管，加入250μL Fragment Buffer(LFB)并轻弹至完全混匀，瞬时离心后重新放置于磁力架上，待液体澄清后去掉上清；
e)重复步骤d)；
f)取下离心管，瞬时离心后重新放置于磁力架上，待液体澄清后用小体积的吸头吸出 残留液体；
g)取下离心管，加入25μL Elution Buffer(EB)轻弹混匀，室温孵育10min；
h)将离心管瞬时离心后，放置于磁力架上，待液体澄清后取出25μL上清于新的1.5mL低吸附离心管中，取1μL测定浓度，计算得率。
(4)利用ONT Premethion平台，进行R 9.4芯片的测序，获得测序结果。
所获得的测序结果如下：
下机数据量：66Gb。
平均插入片段长度：22Kb。
Q值：9.2。
比较第一种方法和第二种方法所获得测序结果，不难看出，采用第一种方法构建测序文库测序，能够显著提高Q值，表明采用第一种方法能够提高ONT平台单碱基准确度。
在本发明的描述中，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (16)

  1. 一种测序文库的构建方法，其特征在于，包括：
    将线性双链DNA样本进行损伤修复和末端修复，以便获得经修复的DNA样本；
    将所述经修复的DNA样本进行同源重组，以便获得环状双链DNA分子；
    以所述环状双链DNA分子变性的环状单链DNA分子为模板，进行滚环扩增，以便获得RCA扩增产物；
    将所述RCA扩增产物与测序接头连接，以便获得所述测序文库。
  2. 根据权利要求1所述的构建方法，其特征在于，利用同源重组酶进行所述同源重组。
  3. 根据权利要求1所述的构建方法，其特征在于，进一步包括：
    在将所述经修复的DNA样本进行同源重组之前，在所述经修复的DNA样本的两端连接寡核苷酸序列。
  4. 根据权利要求3所述的构建方法，其特征在于，所述寡核苷酸序列为双链DNA分子。
  5. 根据权利要求1所述的构建方法，其特征在于，在获得经修复的DNA样本之后，在将所述经修复的DNA样本进行同源重组之前；在获得环状双链DNA分子之后，在以所述环状双链DNA分子变性的环状单链DNA分子为模板，进行滚环扩增之前；在获得所述扩增产物之后，在将所述扩增产物与测序接头连接之前；或者在将所述扩增产物与所述测序接头连接之后，分别进行纯化。
  6. 根据权利要求5所述的构建方法，其特征在于，利用磁珠进行所述纯化。
  7. 根据权利要求1所述的构建方法，其特征在于，利用热稳定的DNA聚合酶进行所述滚环扩增。
  8. 根据权利要求1所述的构建方法，其特征在于，利用BST DNA聚合酶进行所述滚环扩增。
  9. 根据权利要求1所述的构建方法，其特征在于，所述测序文库适合于纳米孔平台测序。
  10. 一种测序方法，其特征在于，所述测序方法包括：
    基于权利要求1～9中任一项所述的构建方法，获得测序文库，
    利用纳米孔测序技术对所述测序文库进行测序。
  11. 一种试剂盒，其特征在于，所述试剂盒包括：
    同源重组酶，
    BST DNA聚合酶，
    寡核苷酸序列。
  12. 根据权利要求11所述的试剂盒，其特征在于，所述试剂盒进一步包括下列中的至少一种：
    末端修复物和/或损伤修复物，
    磁珠，
    测序接头，
    RCA引物，所述RCA引物如SEQ ID NO:2所示。
  13. 根据权利要求11所述的试剂盒，其特征在于，所述寡核苷酸序列为双链DNA分子，所述寡核苷酸序列的一条链如SEQ ID NO:1所示，所述寡核酸序列的另一条链为SEQ ID NO:1的互补序列。
  14. 一种制备环状双链DNA分子的方法，其特征在于，包括：
    提供线性双链DNA分子和寡核苷酸序列；
    将所述寡核苷酸序列连接到所述线性双链DNA分子的两端，获得第一连接产物；
    所述第一连接产物在同源重组酶的作用下生成同源重组拼接产物，即获得所述环状双链DNA。
  15. 根据权利要求14所述的方法，其特征在于，所述同源重组酶为NEBuilder同源重组酶，所述寡核苷酸序列为双链DNA分子，所述寡核苷酸序列的一条链如SEQ ID NO:1所示，所述寡核酸序列的另一条链为SEQ ID NO:1的互补序列。
  16. 同源重组在RCA扩增中的应用，其特征在于，所述应用包括：
    线性双链DNA分子通过同源重组制备得到环状双链DNA分子；
    所述环状双链DNA分子变性的环状单链DNA分子为模板，进行滚环扩增。
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