WO2017215500A1 - 一种核酸等温自扩增方法 - Google Patents

一种核酸等温自扩增方法 Download PDF

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WO2017215500A1
WO2017215500A1 PCT/CN2017/087414 CN2017087414W WO2017215500A1 WO 2017215500 A1 WO2017215500 A1 WO 2017215500A1 CN 2017087414 W CN2017087414 W CN 2017087414W WO 2017215500 A1 WO2017215500 A1 WO 2017215500A1
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陆欣华
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention relates to a method for isothermal self-amplification of nucleic acids, and belongs to the technical field of nucleic acid amplification.
  • nucleic acid amplification technology has revolutionized molecular biology research and pathogenic microbial detection.
  • Isothermal Amplification Technology is an in vitro amplification technique for nucleic acids. The reaction process is always maintained at a constant temperature, and the purpose of rapid nucleic acid amplification is achieved by adding different active enzymes and respective specific primers.
  • Common isothermal amplification techniques are as follows: loop-mediated nucleic acid isothermal amplification technology (LAMP); rolling circle amplification technology RCA; single primer isothermal amplification SPIA; isokinetic amplification technology relying on helicase; HAD; Amplification of SDA, and the like; however, existing methods are generally prone to non-specific amplification due to the presence of a DNA polymerase having strand displacement activity in the presence of a primer.
  • Isothermal amplification technology not only widely used in second-generation nucleic acid sequencing technology, but also plays a very important role in the third-generation nucleic acid sequencing technology, such as the use of rolling ring expansion in the sequencing technology of Pacific Biosciences Increased technology RCA (Patent No.: US9404146), using this technology to repeatedly sequence the same segment, and then through software calibration, greatly improved the accuracy of sequencing data.
  • the accuracy of sequencing data can be greatly improved (INC-Seq: Accurate single molecule reads using nanopore sequencing, Li et al. GigaScience (2016) 5: 34).
  • the rolling circle amplification technology RCA is adopted, which is not only complicated, but also the amplification is not uniform enough, which affects the sequencing results.
  • the object of the present invention is to solve the above problems and to provide a method for isothermal amplification of nucleic acids.
  • a method for isothermal self-amplification of nucleic acid comprising the following steps,
  • the DNA linker of the desired sequence is a linear nucleic acid fragment having a spontaneously formed self-stem loop structure in which no additional amplification primers need to be added;
  • the reagent includes a DNA polymerase having strand displacement activity or another polymerase having strand displacement activity.
  • the DNA polymerase is a Bst enzyme, or another DNA polymerase.
  • the DNA linker added at both ends of the target template is a linear nucleic acid fragment having a palindromic complementary sequence, which is capable of spontaneously forming a self-stem loop structure, thereby triggering DNA polymerase to perform extension amplification.
  • the sequence of the DNA linker may be, but not limited to, a repeat sequence of a base AT combination in an application case, or a repeat sequence of an AATT combination, or a repeat sequence of a GC combination.
  • the DNA linker is a linker that can perform base modification.
  • the modification may be a thio modification or the like.
  • the product of the amplification method is a folded complementary single stranded DNA having a repeat sequence in tandem.
  • An application of a nucleic acid isothermal self-amplification method which can be used for the establishment of a second generation sequencing library, which breaks a long strand of genomic DNA into small fragments, and has a suitable sequence at both ends of the fragment
  • the adaptor is subjected to circular amplification; or the selective insertion of the adaptor fragment by a transposon, a crispr/cas9 system, etc., and the amplification product is a tandem repeat sequence, and the sequencing library prepared by the method is used.
  • Some sequencing machines such as nanopore sequencing instruments, can increase the number of sequencing sequences of interest. Thereby improving the sequencing accuracy.
  • the target fragment can be rapidly and efficiently amplified
  • amplification can be performed independently of the primer
  • the amplified product is a long single-stranded DNA of continuous complementary sequence, which can be applied to special occasions.
  • Amplification has no GC preference.
  • Figure 1 Schematic diagram of the respiratory mechanism and conformational transition process at the DNA double-stranded ends.
  • Figure 2 Schematic representation of the process of self-amplification in the case of DNA double-stranded ends (3' and 5' ends) suitable for self-amplifying sequences.
  • Figure 3 Schematic representation of the process of adding appropriate sequences at the end of the gene of interest.
  • Figure 4 Schematic representation of the process of adding appropriate sequences at the end of a gene of interest using non-specific amplification.
  • Figure 5 Schematic diagram of sequence structure changes during the construction of a second generation sequencing library.
  • the invention discloses a method for isothermal self-amplification of nucleic acids, the product of which is a folded complementary single-stranded DNA.
  • a method for isothermal self-amplification of nucleic acid comprising the following steps,
  • the DNA linker of the desired sequence is linear with spontaneous formation of its own stem-loop structure Nucleic acid fragments, which do not require the addition of additional amplification primers;
  • the DNA linker added at both ends of the target template is a linear nucleic acid fragment having a palindromic complementary sequence, which is capable of spontaneously forming a self-stem loop structure, thereby triggering DNA polymerase to perform extension amplification.
  • the target template can form a self-stem loop structure by adding a suitable palindromic complementary sequence at the linear end of the DNA by PCR, ligase, in vitro transposon system, and the like.
  • the product of the amplification method is a folded complementary single-stranded DNA.
  • the reagent includes a DNA polymerase having strand displacement activity, or other DNA polymerase.
  • the DNA polymerase is a Bst enzyme.
  • the Bst enzyme has no GC preference for amplification, resulting in better homogeneity of the amplified product.
  • An application of a nucleic acid isothermal self-amplification method which can be used for the establishment of a second-generation sequencing library, comprising the steps of: after fragmenting the DNA, adding a sequence suitable for self-amplification at both ends of the DNA by ligase reaction; Alternatively, a selective insert such as a transposon, a crispr/cas9 system, or the like, and a sequence suitable for self-amplification are ligated to the end of the DNA fragment for isothermal self-amplification.
  • the double-stranded DNA ends are in the dynamic equilibrium of melting into free single strands and pairing complementary into double strands.
  • the foreign-derived matching primer can directly invade and bind to one of the DNA strands, and the extension amplification is performed by the DNA polymerase having strand displacement activity (Isothermal amplification method for next-generation sequencing 14320–14323
  • the invention first introduces a special DNA sequence at the end of the target gene fragment by PCR or linker ligation, DNA directed recombination and the like.
  • This sequence is a palindrome sequence. It can be paired by itself to form a stem-loop structure, thereby utilizing DNA respiration phenomenon, without the need to add exogenous primers, and the 3' end can be directly extended by itself as a template under the action of a DNA polymerase having strand displacement activity.
  • the new 3' end and the original 5' end sequence are complementary, and the palindromic sequence can also be formed, and the self-complementary pairing is performed for a new round of extension amplification.
  • the new 3' end is still the original 5' end.
  • the terminal sequences are complementary and cyclically amplified. Each single strand of the DNA double strand carries out a similar reaction at the same time, and the final product is two multiple repeating complementary DNA single strands.
  • the complementary bases in the DNA double strand are linked by hydrogen. Since there are two hydrogen bonds in A (adenine) and T (thymine), there are three hydrogen bonds in G (guanine) and C (cytosine). Therefore, the GC is more difficult to open than the AT. In the phenomenon of DNA respiration, the hairpin structure conformation at the 3' end is formed, and compared with the fully complementary conformation of DNA, not only the terminal pairing bases are less, but also the loop portions cannot be paired due to the spatial structure.
  • the AT content can be increased in the terminal sequence to increase the length of the free single chain, and short repeat sequences such as TA repeats or repeat sequences such as TAA and TTAA are used. Make paired binding easier.
  • the end product of isothermal self-amplification is a single-stranded DNA of contiguous complementary sequence, which can be converted into short fragments by enzymatic cleavage and the like.
  • long repeat fragments can be applied to special occasions such as sequencing.
  • a second generation sequencing library was constructed. Genomic DNA is a long sequence that is interrupted into small fragments by mechanical shearing (such as the addition of magnetic bead oscillations) or by enzymatic cleavage, about a few hundred bp in length, and then ligated to the enzyme.
  • a adaptor sequence is added at the end, which contains sequences suitable for self-amplification, followed by isothermal self-amplification.
  • the amplification process did not use amplification primers, nor did the double-strand fully open step, so the product had better homogeneity and no GC preference than the PCR method.
  • the respiratory mechanism and conformational transition of the DNA double-stranded ends.
  • a linearly complementary DNA double strand (type B) under physiological conditions, the terminal double strand opens into a free state (type A), and the two states are dynamically converted to each other.
  • the breathing is strong.
  • a stem-loop structure type C is generated, and a DNA polymerase binding site appears, triggering the amplification reaction.
  • the DNA double-stranded ends (3' and 5' ends) have a self-amplification process suitable for self-amplifying sequences.
  • the first step double-stranded linear DNA; the second step, the 3' end presents a hairpin structure, the DNA polymerase with strand displacement activity binds, and the strand displacement reaction takes place; the third step completes the amplification of itself, the antisense strand It is replaced (the antisense strand also reacts synchronously), the copy number is doubled, and the newly generated 3' end is modeled by the 5' end, and the hairpin structure can still be presented; in the fourth step, the N round is cyclically amplified.
  • the product after cyclic amplification is a long single-stranded DNA with a copy number of 2 N.
  • a method of adding a suitable sequence to the end of a gene of interest by a PCR method is used.
  • the 3' end of primer A is the complementary sequence of the target gene
  • the 5' end is the added sequence
  • the primer is bound after high temperature melting, and the antisense strand is formed by PCR amplification
  • the second step is the 3' end of primer B.
  • the complementary sequence of the gene, the 5' end is an added sequence, the primer is bound after high temperature melting, and PCR is amplified
  • the third step after the high temperature melting, the amplified product itself is looped, and becomes a starting material capable of isothermal self-amplification.
  • a method of adding a suitable sequence to the end of a gene of interest is carried out by a non-specific amplification method.
  • primer A is bound after high temperature melting
  • DNA polymerase is amplified to form an antisense strand
  • some DNA polymerase having strand displacement activity For example, Bst enzyme can still combine and expand when the primer sequence and template are not completely matched. Usually this is the unfavorable side of the reaction. If this DNA is long enough, the 3' end can be bent.
  • Amplification is carried out by non-specific binding on itself to obtain a complementary sequence at the 5' end; and the fourth step is to be a starting material capable of isothermal self-amplification.
  • sequence is: SEQ ID NO. 2
  • the reaction product was digested with restriction endonuclease Hind III (purchased from NEB) and detected by qPCR.
  • the qPCR reaction system was 20 ul, and the final concentration of the primer was 200 nM, Fast sybgreen Mix 1x, and the product was digested with 3 ⁇ l. Reaction procedure, 95 ° C, 30 seconds, then 45 cycles 60 ° C, 20 Seconds, 95 ° C, 5 seconds.
  • Hind Standard 1 was amplified about 1000 times, and Hind Standard 2 was hardly amplified, indicating that the nucleic acid fragment having a specific structure can be subjected to constant temperature self-amplification.

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Abstract

提供一种核酸等温自扩增方法，所述方法通过在目标模板两端添加合适的回文互补序列，自身自发形成茎环结构；及提供反应所需的试剂和条件进行自扩增，该方法不需要添加额外扩增引物；所述试剂包括具有链置换活性的DNA聚合酶。该方法不依赖于外源扩增引物进行扩增；扩增温度恒定，不需要复杂的温控设备，扩增快速；扩增产物是连续互补序列的长单链DNA，可以应用于特殊场合；扩增没有GC偏好性。

Description

一种核酸等温自扩增方法 技术领域
本发明涉及一种核酸等温自扩增方法，属于核酸扩增技术领域。
背景技术
近几十年来，核酸扩增技术为分子生物研究和病原微生物检测做出了革命性的贡献。
等温扩增技术(Isothermal Amplification Technology)是核酸体外扩增技术，其反应过程始终维持在恒定的温度下，通过添加不同活性的酶和各自特异性引物来达到快速核酸扩增的目的。常见的等温扩增技术有以下几种:环介导核酸等温扩增技术(LAMP)；滚环扩增技术RCA；单引物等温扩增SPIA；依赖解旋酶的等温扩增技术HAD；链替代扩增SDA，等等；但现有的方法由于具有链置换活性的DNA聚合酶在引物存在的情况下，普遍容易产生非特异性扩增。
等温扩增技术，不仅仅在第二代核酸测序技术中广泛使用，在第三代核酸测序技术中也扮演了非常重要的角色，比如在美国Pacific Biosciences公司的测序技术中就运用了滚环扩增技术RCA(专利号：US9404146)，运用这个技术对同一片段反复测序，然后通过软件校准，极大的提高了测序数据的准确性。同样，英国Oxford Nanopore公司的测序技术中，如果使用类似技术，也可以极大的提高测序数据的准确性(INC-Seq:Accurate single molecule reads using nanopore sequencing，Li et al.GigaScience(2016)5:34)。但由于英国Oxford Nanopore公司的测序原理比较特殊，采用了滚环扩增技术RCA，不仅仅步骤繁杂，而且扩增不够均匀，影响测序结果。
发明内容
本发明的目的在于解决上述问题，提出一种核酸等温自扩增的方法。
本发明的目的通过以下技术方案来实现：
一种核酸等温自扩增方法，包括如下步骤，
a、在目标模板两端添加所需序列的DNA接头；
c、提供反应所需的试剂和条件；
所述所需序列的DNA接头为具有自发形成自身茎环结构的线性核酸片段，所述自扩增方法中不需要添加额外扩增引物；
所述试剂包括具有链置换活性的DNA聚合酶或其他具有链置换活性的聚合酶。
优选地，所述DNA聚合酶为Bst酶，或者其他DNA聚合酶。
优选地，所述目标模板两端添加的DNA接头为具有回文互补序列的线性核酸片段，能够自发形成自身茎环结构，从而触发DNA聚合酶进行延伸扩增。
优选地，所述DNA接头的序列可以是但不限于，应用例中碱基AT组合的重复序列，或者是AATT组合的重复序列，或GC组合的重复序列。所述DNA接头为可以进行碱基修饰的接头。所述修饰可以为硫代修饰等等。
优选地，所述扩增方法的产物为折叠互补单链DNA，具有串联的重复序列。
一种核酸等温自扩增方法的应用，所述方法可用于二代测序文库的建立，所述文库建立方法通过将基因组DNA长链打断成小片段，在片段两端连接上含有适合序列的接头，进行循环扩增；或者通过转座子，crispr/cas9系统等选择性的插入接头片段，进行选择性扩增，扩增产物是串联的重复序列，以这种方法制备的测序文库，使用某些测序机器，比如纳米孔测序仪器，可以增加对目的序列的测序次数， 从而提高测序精度。
本发明的有益效果主要体现在：
1、在等温情况下，可以对目的片段快速有效扩增；
2、在DNA末端引入合适的回文互补序列后，可以不依赖于引物进行扩增；
3、扩增产物是连续互补序列的长单链DNA，可以应用于特殊场合。
4、扩增没有GC偏好性。
附图说明
下面结合附图对本发明技术方案作进一步说明：
图1：DNA双链末端的呼吸机制和构象转换过程示意图。
图2：DNA双链末端(3＇端和5＇端)均有适合自扩增序列的情况下，自扩增的过程示意图。
图3:在目的基因末端添加合适序列的过程示意图。
图4:采用非特异性扩增法在目的基因末端添加合适序列的过程示意图。
图5:二代测序文库构建过程中的序列结构变化示意图。
具体实施方式
本发明揭示了一种核酸等温自扩增方法，所述扩增方法的产物为折叠互补单链DNA。
包括如下步骤，
一种核酸等温自扩增方法，包括如下步骤，
a、在目标模板两端添加所需序列的DNA接头；
c、提供反应所需的试剂和条件；
所述所需序列的DNA接头为具有自发形成自身茎环结构的线性 核酸片段，所述自扩增方法中不需要添加额外扩增引物；
具体的，所述目标模板两端添加的DNA接头为具有回文互补序列的线性核酸片段，能够自发形成自身茎环结构，从而触发DNA聚合酶进行延伸扩增。
所述目标模板通过PCR，连接酶，体外转座子系统等，在DNA线性末端添加合适的回文互补序列，从而能够形成自身茎环结构。
本发明中所述自扩增方法中不需要添加额外扩增引物；所述扩增方法的产物为折叠互补单链DNA。
所述试剂包括具有链置换活性的DNA聚合酶，或者其他DNA聚合酶。
所述DNA聚合酶为以Bst酶。Bst酶对扩增没有GC偏好，使得扩增产物具有更好的均一性。
一种核酸等温自扩增方法的应用，所述方法可用于二代测序文库的建立，包括如下步骤，将DNA片段化后，通过连接酶反应，在DNA两端添加适合自扩增的序列；或者通过转座子，crispr/cas9系统等选择性的插入片段，最终在DNA片段末端连接有适合自扩增的序列，进行等温自扩增。
以下具体阐述下本发明的自扩增原理：
根据DNA呼吸机制，双链DNA末端处在解链成游离单链和配对互补成双链的动态平衡中，温度升高，呼吸作用加剧，末端双链解开越显著。此时外源的匹配引物可以直接入侵和DNA其中的一条链结合，在具有链置换活性的DNA聚合酶作用下，延伸扩增(Isothermal amplification method for next-generation sequencing 14320–14323|PNAS|August 27,2013|vol.110|no.35)
本发明首先通过PCR或者接头连接，DNA定向重组等技术，在目的基因片段末端引入特殊的DNA序列。这段序列是一段回文序列， 可以自身配对，形成茎环结构，从而利用DNA呼吸现象，不需要再添加外源引物，3＇端在具有链置换活性的DNA聚合酶作用下，可以直接以自身为模版进行延伸扩增，第一轮扩增完成后，新的3＇端和原5＇端序列互补，同样可以形成回文序列，自身互补配对，进行新的一轮延伸扩增，新的3＇端仍然和原5＇端序列互补，循环扩增。DNA双链的每条单链同时进行类似反应，最后产物是两条多重复的互补DNA单链。
DNA双链中的互补碱基是通过氢健连接的，由于A(腺嘌呤)与T(胸腺嘧啶)中有两个氢键，G(鸟嘌呤)与C(胞嘧啶)中有三个氢键，所以GC配对比AT更不易打开。在DNA呼吸现象中，要形成3＇端的发夹结构构象，和DNA完全互补的构象相比，不仅末端配对碱基更少，还有环状部分由于空间结构的原因不能配对结合。所以为了增加3＇端成功形成发夹结构的概率，可以在末端序列中增加AT含量，使游离出来的单链长度增加，同时采用短重复序列，比如，TA重复或者TAA，TTAA等重复序列，使配对结合更容易发生。
等温自扩增过程中，一旦在目的片段末端引入合适的序列，即使移除引物后，仍然可以进行自我扩增，而且在扩增的过程中，扩增产物是连续互补的序列，由于空间上的近距离效应，可以迅速配对形成双链结构，几乎没有裸露的单链DNA暴露在外，基本消除非特异性扩增。在高通量测序中，比如单细胞测序，利用等温自扩增，和PCR方法相比，不仅可以消除GC偏好，而且可以有更好的均一性。
等温自扩增的终产物是连续互补序列的单链DNA，一方面可以通过酶切等手段，转换成短片段，另外一方面这种长重复片段，可以应用在特殊场合，比如测序等。如图5所示，二代测序文库构建。基因组DNA是长序列的，通过机械剪切(比如添加磁珠震荡)，或者酶切等技术，打断成小片段，约几百bp长度，然后连接酶作用，在 末端添加接头序列，其中包含适合自扩增的序列，然后等温自扩增。扩增过程没有使用扩增引物，也没有双链完全打开的步骤，所以和PCR方法相比，产物具有更好的均一性和没有GC偏好性。
以下结合附图具体阐述本发明通过不同的方法产生茎环结构以适用自扩增反应。
如图1所示，DNA双链末端的呼吸机制和构象转换。线性完全互补的DNA双链(B型)，生理条件下，末端双链打开成游离状态(A型)，两种状态互相转换动态平衡。在高温或其他条件下，呼吸强烈，当末端序列自身互补的时候，会产生茎环结构(C型)，出现DNA聚合酶的结合位点，触发扩增反应
如图2所示，DNA双链末端(3＇和5＇端)均有适合自扩增序列的情况下，自扩增的过程。第一步，双链线性DNA；第二步，3＇端呈现发夹结构，具有链置换活性的DNA聚合酶结合，发生链置换反应；第三步，完成对自身的扩增，反义链被置换下来(反义链也同步反应)，拷贝数增加一倍，新产生的3＇端都是以5＇端为模版，仍然可以呈现发夹结构；第四步，循环扩增N轮。第五步，循环扩增后产物是一条长单链DNA，拷贝数为2的N次方。
如图3所示，采用PCR方法在目的基因末端添加合适序列的方法。第一步，引物A的3＇端为目的基因互补序列，5＇端为添加序列，高温解链后引物结合，PCR扩增形成反义链；第二步，引物B的3＇端为目的基因互补序列，5＇端为添加序列，高温解链后引物结合，PCR扩增；第三步，高温解链后扩增产物自身成环，成为可以进行等温自扩增的起始物。
如图4所示，采用非特异性扩增法进行在目的基因末端添加合适序列的方法。第一步，高温解链后引物A结合；第二步，DNA聚合酶扩增形成反义链；第三步，有些具有链置换活性的DNA聚合酶， 比如Bst酶，在引物序列和模版不完全匹配的情况下，仍然可以结合并进行扩增，通常这是对反应不利的一面，利用这一特性，如果DNA足够长，3＇端弯曲后能够在自身上非特异性结合进行扩增，从而得到5＇端的互补序列；第四步：成为可以进行等温自扩增的起始物。
扩增效率检测：
为了排除过量引物，模版核酸等干扰，更好的验证DNA等温自扩增效率，人工合成一段末端具有等温自扩增序列的单链DNA，命名Hind标准品1，序列为SEQ ID NO.1(以下所有DNA均从生工生物工程(上海)股份有限公司合成)。
Figure PCTCN2017087414-appb-000001
同时人工合成了一段末端不具备等温自扩增序列的单链DNA(去掉了末端互补序列)命名Hind标准品2。
序列为：SEQ ID NO.2
Figure PCTCN2017087414-appb-000002
恒温扩增反应体系25μl，其中dNTP 0.2mM each、Bst DNA Polymerase Buffer1×、4mM MgSO4、warm strat Bst DNA Polymerase 8U(购自NEB公司),Hind标准品约100pM，60℃反应1小时。
反应产物用限制性内切酶Hind III(购自NEB公司)酶切后，qPCR检测。
qPCR前引物序列：CGCGCGTAGCAGCACGTAAATA
qPCR后引物序列：GTGCAGGGTCCGAGGT
qPCR反应体系20ul，含引物终浓度200nM，Fast sybgreen Mix 1x，酶切产物3μl。反应程序，95℃、30秒，然后45个循环60℃、20 秒，95℃、5秒。
检测数据如表1所示：
表1
Figure PCTCN2017087414-appb-000003
结果显示：Hind标准品1扩增了约1000倍，Hind标准品2几乎没有扩增，说明具有特定结构的核酸片段能够进行恒温自扩增。
扩增效率和起始浓度关系检测：
同样为了排除过量引物，模版核酸等的干扰，更好的验证DNA等温自扩增，选择人工合成片段Hind标准品1，采用不同的起始浓度。
等温扩增和qPCR实验步骤同实施例1中描述，60℃下反应2小时。
数据如表2所示：
表2
Figure PCTCN2017087414-appb-000004
表2
1 500pM 19 7 -12
2 50pM 23 9 -14
3 5pM 26 12 -14
4 500fM 29 16 -13
5 50fM 31 19 -12
6 5fM 31 24 -7
7 0.5fM 31 27 -4
8 0.05fM 31 30 -1
9 0.005fM 31 31 0
10 0 31 31 0
结果显示：即使在溶液中核酸片段接近单拷贝的时候，仍然能够进行扩增，效率约1000倍，进一步说明，等温自扩增是自发进行的，不需要添加扩增引物。
扩增效率和末端序列关系：
同样为了排除过量引物，模版核酸等的干扰，更好的验证DNA等温自扩增，选择人工合成片段，采用不同长度的末端序列：
Hind标准品1：
Figure PCTCN2017087414-appb-000005
Figure PCTCN2017087414-appb-000006
Hind标准品21:
Figure PCTCN2017087414-appb-000007
Hind标准品15:
Figure PCTCN2017087414-appb-000008
Hind标准品9：
Figure PCTCN2017087414-appb-000009
Hind标准品0：
Figure PCTCN2017087414-appb-000010
等温扩增和qPCR实验步骤同实施例1中描述，60℃反应2小时。
数据如表3所示，
表3：
Figure PCTCN2017087414-appb-000011
Figure PCTCN2017087414-appb-000012
结果显示：末端TA重复序列长度分别为30，21，15的时候，扩增效率约1000倍，长度为9的时候，扩增效率约100倍，长度为0的时候，几乎没有扩增。说明等温自扩增是末端TA重复序列依赖性的。
扩增倍数和时间关系
为了排除过量引物，模版核酸等干扰，更好的验证DNA等温自扩增效率，人工合成一段末端具有等温自扩增序列的单链DNA，Hind标准品1。
等温扩增和qPCR实验步骤同实施例1中描述，60℃反应不同时间。
数据如表4所示，
表4：
  时间(分钟) Ct值(等温扩增酶切后qPCR)
1 0 28
2 1 26
3 5 23
4 20 20
5 60 17
结果显示：等温自扩增时间越长，扩增倍数越高，起始阶段，扩增效率略高。
本发明尚有多种具体的实施方式，凡采用等同替换或者等效变换而形成的所有技术方案，均落在本发明要求保护的范围之内。

Claims (6)

  1. 一种核酸等温自扩增方法，其特征在于：包括如下步骤，
    a、在目标模板两端添加所需序列的DNA接头；
    c、提供反应所需的试剂和条件；
    所述所需序列的DNA接头为具有自发形成自身茎环结构的线性核酸片段，所述自扩增方法中不需要添加额外扩增引物；
    所述试剂包括具有链置换活性的DNA聚合酶或其他具有链置换活性的聚合酶。
  2. 根据权利要求1所述的一种核酸等温自扩增方法，其特征在于：所述DNA聚合酶为Bst酶，或者其他DNA聚合酶。
  3. 根据权利要求1所述的一种核酸等温自扩增方法，其特征在于：所述目标模板两端添加的DNA接头为具有回文互补序列的线性核酸片段，能够自发形成自身茎环结构，从而触发DNA聚合酶进行延伸扩增。
  4. 根据权利要求3所述的一种核酸等温自扩增方法，其特征在于：所述DNA接头的序列可以是但不限于，应用例中碱基AT组合的重复序列，或者是AATT组合的重复序列，或GC组合的重复序列，所述DNA接头为可以进行碱基修饰的接头。
  5. 根据权利要求1所述的一种核酸等温自扩增方法，其特征在于：所述扩增方法的产物为折叠互补单链DNA，具有串联的重复序列。
  6. 一种核酸等温自扩增方法的应用，其特征在于：所述方法可用于二代测序文库的建立，所述文库建立方法通过将基因组DNA长链打断成小片段，在片段两端连接上含有适合序列的接头，进行循环扩增；或者通过转座子，crispr/cas9系统等选择性的插入接头片段，进行选择性扩增，所述扩增产物是串联的重复序列。
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10851409B2 (en) 2011-07-25 2020-12-01 Oxford Nanopore Technologies Ltd. Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
CN112980928A (zh) * 2021-04-02 2021-06-18 川北医学院附属医院 一种茎环引物辅助的等温核酸扩增方法
JP2021528090A (ja) * 2018-06-26 2021-10-21 マサチューセッツ インスティテュート オブ テクノロジー Ｃｒｉｓｐｒエフェクター系に基づく増幅法、システム、及び診断法
US11186857B2 (en) 2013-08-16 2021-11-30 Oxford Nanopore Technologies Plc Polynucleotide modification methods
US11352664B2 (en) 2009-01-30 2022-06-07 Oxford Nanopore Technologies Plc Adaptors for nucleic acid constructs in transmembrane sequencing
US11390904B2 (en) 2014-10-14 2022-07-19 Oxford Nanopore Technologies Plc Nanopore-based method and double stranded nucleic acid construct therefor
US11542551B2 (en) 2014-02-21 2023-01-03 Oxford Nanopore Technologies Plc Sample preparation method
US11560589B2 (en) 2013-03-08 2023-01-24 Oxford Nanopore Technologies Plc Enzyme stalling method
US11649480B2 (en) 2016-05-25 2023-05-16 Oxford Nanopore Technologies Plc Method for modifying a template double stranded polynucleotide
US11725205B2 (en) 2018-05-14 2023-08-15 Oxford Nanopore Technologies Plc Methods and polynucleotides for amplifying a target polynucleotide

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11155860B2 (en) 2012-07-19 2021-10-26 Oxford Nanopore Technologies Ltd. SSB method
CN108588182B (zh) * 2018-04-13 2025-11-28 武汉中科先进技术研究院有限公司 基于crispr-链取代的等温扩增及检测技术
CN109055499B (zh) * 2018-08-30 2021-01-19 杭州杰毅生物技术有限公司 基于CRISPR-Cas的等温核酸检测方法及试剂盒
CN110607355A (zh) * 2019-02-18 2019-12-24 华东理工大学 一种基于Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法及试剂盒
CN112646862B (zh) * 2020-12-28 2022-07-15 天津市疾病预防控制中心 一种新型的自我链置换原理及其多重链置换反应的应用
CN113249438B (zh) * 2021-04-29 2022-04-19 北京擎科生物科技有限公司 一种dna分子量标准片段扩增单链引物、扩增方法以及dna分子量标准的制备方法
WO2026050948A1 (zh) * 2024-09-04 2026-03-12 深圳华大生命科学研究院 核酸文库的构建方法及应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101415838A (zh) * 2003-09-26 2009-04-22 首科安普有限公司 用滚环扩增法扩增多核苷酸
CN102084001A (zh) * 2008-03-28 2011-06-01 加利福尼亚太平洋生物科学股份有限公司 用于核酸测序的组合物和方法
WO2013096802A1 (en) * 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Amplification primers and methods
WO2016022557A1 (en) * 2014-08-05 2016-02-11 Twist Bioscience Corporation Cell free cloning of nucleic acids
WO2016028887A1 (en) * 2014-08-19 2016-02-25 Pacific Biosciences Of California, Inc. Compositions and methods for enrichment of nucleic acids
CN105442054A (zh) * 2015-11-19 2016-03-30 杭州谷坤生物技术有限公司 对血浆游离dna进行多目标位点扩增建库的方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7368265B2 (en) * 2006-01-23 2008-05-06 Compass Genetics, Llc Selective genome amplification
GB0910302D0 (en) 2009-06-15 2009-07-29 Lumora Ltd Nucleic acid amplification
US20120196279A1 (en) * 2011-02-02 2012-08-02 Pacific Biosciences Of California, Inc. Methods and compositions for nucleic acid sample preparation
CN103146684A (zh) * 2013-03-05 2013-06-12 许昌学院 一种等温扩增核酸片段的方法
CN104164488B (zh) * 2014-07-09 2016-11-02 青岛艾菲生物技术有限公司 一种单引物引发的核酸恒温扩增方法
GB201418159D0 (en) 2014-10-14 2014-11-26 Oxford Nanopore Tech Ltd Method
CN104862400B (zh) * 2015-05-25 2019-01-15 浙江大学 一种基于定位探针介导剪切的核酸等温扩增方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101415838A (zh) * 2003-09-26 2009-04-22 首科安普有限公司 用滚环扩增法扩增多核苷酸
CN102084001A (zh) * 2008-03-28 2011-06-01 加利福尼亚太平洋生物科学股份有限公司 用于核酸测序的组合物和方法
WO2013096802A1 (en) * 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Amplification primers and methods
WO2016022557A1 (en) * 2014-08-05 2016-02-11 Twist Bioscience Corporation Cell free cloning of nucleic acids
WO2016028887A1 (en) * 2014-08-19 2016-02-25 Pacific Biosciences Of California, Inc. Compositions and methods for enrichment of nucleic acids
CN105442054A (zh) * 2015-11-19 2016-03-30 杭州谷坤生物技术有限公司 对血浆游离dna进行多目标位点扩增建库的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP3470529A4 *
TRAVERS, K.J. ET AL.: "A Flexible and Efficient Template Format for Circular Consensus Sequencing and SNP Detection", NUCLEIC ACIDS RESEARCH, vol. 38, no. 15, 22 June 2010 (2010-06-22), pages e159, XP002615293 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11352664B2 (en) 2009-01-30 2022-06-07 Oxford Nanopore Technologies Plc Adaptors for nucleic acid constructs in transmembrane sequencing
US11459606B2 (en) 2009-01-30 2022-10-04 Oxford Nanopore Technologies Plc Adaptors for nucleic acid constructs in transmembrane sequencing
US11168363B2 (en) 2011-07-25 2021-11-09 Oxford Nanopore Technologies Ltd. Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
US10851409B2 (en) 2011-07-25 2020-12-01 Oxford Nanopore Technologies Ltd. Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
US11261487B2 (en) 2011-07-25 2022-03-01 Oxford Nanopore Technologies Plc Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
US12168799B2 (en) 2011-07-25 2024-12-17 Oxford Nanopore Technologies Plc Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
US11560589B2 (en) 2013-03-08 2023-01-24 Oxford Nanopore Technologies Plc Enzyme stalling method
US11186857B2 (en) 2013-08-16 2021-11-30 Oxford Nanopore Technologies Plc Polynucleotide modification methods
US11542551B2 (en) 2014-02-21 2023-01-03 Oxford Nanopore Technologies Plc Sample preparation method
US12584168B2 (en) 2014-02-21 2026-03-24 Oxford Nanopore Technologies Plc Sample preparation method
US11390904B2 (en) 2014-10-14 2022-07-19 Oxford Nanopore Technologies Plc Nanopore-based method and double stranded nucleic acid construct therefor
US11649480B2 (en) 2016-05-25 2023-05-16 Oxford Nanopore Technologies Plc Method for modifying a template double stranded polynucleotide
US11725205B2 (en) 2018-05-14 2023-08-15 Oxford Nanopore Technologies Plc Methods and polynucleotides for amplifying a target polynucleotide
JP2021528090A (ja) * 2018-06-26 2021-10-21 マサチューセッツ インスティテュート オブ テクノロジー Ｃｒｉｓｐｒエフェクター系に基づく増幅法、システム、及び診断法
CN112980928A (zh) * 2021-04-02 2021-06-18 川北医学院附属医院 一种茎环引物辅助的等温核酸扩增方法

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CN107488656B (zh) 2020-07-17
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