WO2020034746A1 - 核酸的恒温扩增引物及其应用 - Google Patents

核酸的恒温扩增引物及其应用 Download PDF

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WO2020034746A1
WO2020034746A1 PCT/CN2019/091694 CN2019091694W WO2020034746A1 WO 2020034746 A1 WO2020034746 A1 WO 2020034746A1 CN 2019091694 W CN2019091694 W CN 2019091694W WO 2020034746 A1 WO2020034746 A1 WO 2020034746A1
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primer
amplification
sequence
nucleic acid
strand
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French (fr)
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陆欣华
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陆欣华
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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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

Definitions

  • the invention relates to the technical field of molecular biology, in particular to a constant temperature amplification primer for nucleic acid and application thereof.
  • Nucleic acid amplification technology is widely used in the field of nucleic acid detection, including nucleic acid sequence determination.
  • Conventional nucleic acid amplification technologies include PCR (polymerase chain reaction) technology, as well as isothermal amplification technology RPA (recombinase polymerase amplification), HAD (helicase-dependent nucleic acid isothermal amplification technology), etc., these technologies can Amplify the amount of nucleic acid quickly, but cannot change the length of the nucleic acid, and cannot produce long-chain nucleic acid with end-to-end tandem structure.
  • nucleic acid amplification technologies such as RCA (Rolling Circle Amplification), LAMP (Loop-mediated Isothermal Amplification), etc., can produce long-chain nucleic acids, but the reaction cannot be controlled, and the repeats in the tandem structure cannot be fine-tuned. Number of fragments.
  • nucleic acid detection only needs to amplify the amount of nucleic acid, in some special occasions, it is very useful to generate repeated tandem structures.
  • sequencing Accuracy is limited.
  • ONT Olford Nanopore ONT
  • PacBio's sequencing instruments have a single nucleic acid sequencing accuracy of about 90%, which cannot meet the special needs applications.
  • PacBio Pacific Biological Corporation
  • CCS Cyclic Alignment Sequencing
  • the ONT company sequenced the same DNA double strand separately, it obtained two repeats, and after software calibration, it could significantly improve the accuracy rate to about 96%.
  • Some laboratories have used special amplification methods to generate more repeated tandem structures and have achieved higher accuracy. For example, INC-Seq: accurate single molecule readings using nanopore sequencing (DOI 10.1186 / s13742-016- 0140-7) and so on, but these methods have many steps, long time, high cost, and difficult to popularize. Therefore, the existing amplification technologies either cannot produce long-chain nucleic acids with a tandem structure or the length of the nucleic acids cannot be precisely controlled.
  • sequencing accuracy can be improved by repeatedly sequencing the same sequence, but the output throughput of the instrument itself needs to be consumed. If the measurement is repeated twice, the final output data will be reduced by about half. If the sequencing is repeated 10 times The output data will be reduced by about 90%. It is usually the most cost-effective mode to repeat the test twice. It is reported that there is a significant marginal diminishing effect on the improvement of accuracy after repeated test 6 times. Therefore, if the number of repetitions in the amplified fragment can be controlled reasonably, and the amplification step is simple and fast, it will have important practical significance.
  • the object of the present invention is to provide a thermostatic amplification primer for nucleic acid and its application.
  • a nucleic acid isothermal amplification primer includes a front primer and a rear primer for amplifying a gene of interest, and the front primer is a sequence having an exogenous self-paired hairpin structure at the 5 ′ end, or The 5 'end has a sequence of a hairpin structure formed by matching any source sequence in the target gene close to the front primer end.
  • the back primer is the same as the downstream primer sequence required when completing the amplification of the target gene or the back primer is the same sequence as the downstream primer sequence required when completing the amplification of the target gene and its downstream primer
  • the 5 'sequence consists of a sequence of exogenous self-pairing hairpin structures.
  • the method is applied to a method for isothermal nucleic acid amplification that can produce a tandem repeat sequence of an amplified gene of interest.
  • the application method includes, but is not limited to, an RPA amplification method, a HAD amplification method, and a PCR amplification method.
  • the amplified product is applied to library preparation of a sequencing instrument.
  • a nucleic acid amplification is performed by upstream primers and downstream primers to form a DNA target fragment, and the target fragment is composed of a sense strand and an antisense strand;
  • the front primer is a sequence having an exogenous self-pairing hairpin structure at the 5 ′ end, or having a sequence with the target at the 5 ′ end.
  • a sequence of a hairpin structure formed by matching any source sequence in the target primer end near the front primer end;
  • Single primer amplification is performed by adding the primers again to form a desired amplification product having a structure in which a sense strand and an antisense strand are connected in series on the same strand.
  • the S2 is formed at one end to introduce an amplification product that can form a hairpin loop sequence, and the S3 is formed to have the same sense strand and antisense strand. Diploid amplification products with a single tandem structure on one strand;
  • the S2 forms a diploid amplification having a structure in which the sense strand and the antisense strand are connected in series on the same strand once.
  • the S3 forms a tetraploid amplification product having a structure in which the sense strand and the antisense strand are tandem twice on the same strand.
  • the single primer amplification step in S3 is repeated N times, and the S3 formation is
  • the S3 formation is
  • tetraploid amplification products that are structured in series twice on the same strand with the antisense strand
  • polyploidy products will be formed with the sense strand and the antisense strand repeated in series on the same strand multiple times.
  • the product of ploidy amplification, each of the polyploids increases exponentially, and the ratio of the number of each of the polyploids will increase as the number of repetitions increases, and the N ⁇ 1.
  • the products produced after repeating S3, in addition to the 4-ploid amplification products, the products produced will also be accompanied by the production of 8- and 16-ploid-assisted amplification products; when it is repeated again, the repeat, The ratio of the 8-ploid and 16-ploid will be greatly increased, and at the same time, the amplification products of 32- and 64-ploids are also generated, and so on, the proportion of the amplified products is limited by the amplification efficiency There are no explicit restrictions here.
  • the method for thermostatic amplification of nucleic acids by the primers generating tandem repeat sequences further comprises the following steps,
  • the method for introducing a differential sequence in S4 includes, but is not limited to, attaching a Y-type adapter through a ligation reaction, and the differential sequence is a new primer in S4 amplification.
  • the outstanding effects of the present invention are as follows: 1.
  • the nucleic acid amplification technology using the primers of the present invention produces a structure in which the sequence of the target gene is repeated in tandem, and the number of repetitions can be finely controlled, such as 2 times, 4 times, 8 times, etc .
  • the amplification step is not only simple and fast, but also improves the accuracy of sequencing.
  • ONT's sequencing equipment uses modified primers for amplification, and the amplified products can be directly combined with the sequencing primers by chemical methods, eliminating the need for a ligation reaction step, reducing the existing time-consuming 60 minutes to 5 minutes, greatly improving Out of efficiency.
  • Figure 1 Electrophoretic gel maps of the amplification products introduced in the RPA technique mentioned in the present invention.
  • FIG. 2 is a schematic diagram of a step of performing nucleic acid amplification using the primer of the present invention, wherein the front primer is a primer having a hairpin structure.
  • FIG. 3 is a schematic diagram of a step of nucleic acid amplification using the primers of the present invention, wherein the front and rear primers are primers each having a hairpin structure.
  • Figure 4 A schematic diagram of the steps for amplifying a polyploid-based amplification product repeated in tandem twice.
  • Figure 5 Sequencing read length diagram of Example 1 of the present invention.
  • Figure 6 Sequencing read length diagram of Example 2 of the present invention.
  • the invention discloses a constant-temperature amplification primer for nucleic acid and application thereof.
  • the primer includes a front primer and a rear primer for amplifying a target gene, and the front primer is an exogenous self-pairing at the 5 'end.
  • the post primer is the same as the downstream primer sequence required to complete the amplification of the target gene, or the same sequence as the downstream primer sequence required to complete the amplification of the target gene, and it has an exogenous 5 ′ to the downstream primer.
  • Nucleic acid isothermal amplification primers can be applied to RPA amplification methods, HAD amplification methods, and PCR amplification methods to generate amplification products that perform tandem repeats of a target gene.
  • the temperature of the template DNA double-strand is opened by increasing the temperature to 95 ° C in each cycle, and the temperature is reduced to 60 ° C.
  • the primer is bound to the matching portion of the template DNA (deoxyribonucleic acid).
  • the recombinase will scan the double strand of the template DNA with the primer and find the complementarity.
  • a substitution reaction occurs, the primers are bound together, and at the same time, a DNA polymerase capable of strand displacement begins to synthesize new DNA.
  • the target DNA fragment is amplified to form a product of a tandem structure of a sense strand and an antisense strand.
  • the DNA target fragment (Target A + is the sense strand and Target A- is the antisense strand) is an amplified fragment amplified from the front primer (Primer F) and the back primer (Primer R).
  • Step 1 Add the rear primer and the new front primer (Primer LoopA + F) at the same time for the RPA reaction.
  • the sequence of the 3 'end of the new front primer is consistent with that of the Primer F.
  • the 5' end sequence can form a hairpin-shaped loop. .
  • the amplification product of step 1 introduces a sequence that can form a hairpin loop at one end.
  • the second step only the rear primer is added while performing the RPA reaction, and a single primer amplification is performed.
  • the amplification reaction uses Target A + as a template to continuously generate Target A-.
  • the newly generated Target A- can form a hairpin loop at the 3 ′ end and can continue to synthesize using itself as a template.
  • the product is a self-paired, semi-closed, single-stranded DNA.
  • step four the rear primers continue to participate in the RPA reaction.
  • the new amplification product is a double-stranded target sequence at the 5 'end and a target A + sequence at the 3' end, which is about twice the length of the target fragment.
  • the primer can have a reverse complementary sequence at the 5 'end or a sequence at the 5' end. This sequence can be reversed from a part of the sequence near the 5 'end inside the amplicon. To complement each other.
  • the target fragment can be amplified into the amplification product of the sense strand tandem antisense strand, and the amplified product can be regarded as a new target fragment.
  • the amplified product can be regarded as a new target fragment.
  • new similar sequences on both sides For example, the ligation reaction and the addition of a new adaptor sequence
  • an amplification product of the nth power of the repeat 2 can be amplified, where n is an arbitrary integer. In practice, n is limited by the amplification efficiency.
  • primers before and after amplification that have a hairpin structure can also be used.
  • the 5 'end of the primer is a closed circular structure, it has already been paired. Once the complementary structure of the 3' end of the complementary strand is exposed, it can only pair itself and then use itself as a template for amplification. , Forming a sense strand tandem antisense strand, semi-closed, single-stranded DNA. At the same time, the new primers extend the new strand that is generated. After being replaced and freed, the 3 'end will also pair itself, and use itself as a template to amplify, forming a sense strand, a tandem antisense strand, a semi-closed, and a single DNA strand.
  • the amplified product is directly connected to the sense strand and the antisense strand, a semi-closed, and a single DNA strand (a diploid, a new DNA fragment formed by concatenating the sense strand and the antisense strand of the target DNA fragment once). Because the diploid product can also be amplified, there will also be longer amplification products, such as the 4-ploid (a new DNA fragment formed by repeating the sense and antisense strands of the target DNA fragment in tandem).
  • the starting fragment is the amplification product of Figure 2, mainly the diploid of the sense strand tandem antisense strand. If the reaction is complete, continue to add amplified primers with a hairpin structure to perform a single primer Amplification can continue to amplify the 4-ploid-based amplification products. It is also possible to directly use the post-primer with a hairpin structure in the step shown in FIG. 2. After the amplification is completed, the product is mainly a tetraploid. Similarly, the amplification product of FIG. 3 can be amplified by single primer using the pre- or post-amplification primer with a hairpin structure again. It can also continue to amplify the tetraploid-based amplification repeated twice in series. product.
  • HDA This technical solution can also be implemented by the HDA method.
  • HDA's amplification process can be subdivided into six stages: helicase to dissolve double-stranded DNA, single-stranded binding protein and maintain single-stranded, primer-bound, polymerase-bound, catalyzed target sequence synthesis, and helicase-to-new strand recycling.
  • the helicase binds to double-stranded DNA with the help of a helper protein, and begins to decompose the double-stranded DNA into single-stranded DNA with the participation of ATP.
  • the single-stranded DNA that appears is immediately bound by the single-stranded binding protein in the system, stabilizing the single strand of DNA that has just been untied, and preventing double-strands from forming again.
  • the primers then bind to the target sequence.
  • the polymerase binds and catalyzes the synthesis of the target sequence.
  • the newly synthesized sequence enters a new cycle of melting and amplification. After continuous cycling, the target sequence is amplified exponentially, and the products are double-stranded target DNA just like the products of conventional PCR.
  • primers and steps similar to RPA can also be used to amplify the sense strand tandem antisense strand one or more times.
  • the double-strand dynamic separation and binding will occur.
  • the 3 'end has a self-complementary palindrome sequence, there is a certain probability that the sense and antisense strands will completely match when the double strands are separated and re-paired, and there is also a certain probability that they can be found on each strand.
  • a stem-loop structure is formed, and the stem-loop structure at the 3 'end can trigger a new amplification, using itself as a template, and doubling the length.
  • the target DNA fragment has sequences on both sides that can form a stem-loop structure, similar to the amplification product in Figure 3, then without the primer, the target fragment can continue to grow spontaneously and amplify the sense strand tandem antisense. Amplified fragments with multiple strand repeats. The number of repetitions is related to the response time.
  • the reaction can be controlled exactly like the RPA reaction. If the amplification primer uses a stem-loop structure, The reaction can still increase in length when the primers are depleted, and as the amplification time increases, more repetitions occur.
  • This example is used to illustrate the use of hairpin-like primers to amplify a long DNA fragment of the sense strand and the antisense strand of the target gene through a two-step RPA reaction.
  • a 276 bp amplicon was amplified from the Lambda DNA standard as a target fragment, the sequence is:
  • a new front primer 5'-CGTAGCTGATGGCACATATGTGCCATCAGCTACGCCACTTCAGCACGAGATGCGGTGGC-3 'and a back primer were used to perform an RPA reaction on the target fragment.
  • RPA reagent was purchased from the British company TwistDX and was named TwistAmp Basic reagent.
  • the first reaction system is 50ul, containing TwistAmp Basic powder, 2.4ul of pre-amplification primer (10uM), 2.4ul of post-amplification primer (10uM), 29.5ul of reaction buffer, 1ul of target fragment (about 1nM) , 2.5ul of magnesium acetate (280mM), 12.2ul of water. Reaction conditions: 37 ° C, 40 minutes.
  • the second reaction system is 50ul, containing TwistAmp Basic powder, 4.8ul amplified primers (10uM), 29.5ul reaction buffer, 1ul 100 times dilution of the first amplification product, 2.5ul magnesium acetate ( 280mM), 12.2ul of water. Reaction conditions: 37 ° C, 40 minutes.
  • the final amplified product was detected by electrophoresis with bright bands at about 300bp and 600bp.
  • Three-generation sequencing of the amplified products (ONT instrument).
  • the ligation method was used to detect the amplified products.
  • the length was concentrated at 600bp.
  • the abscissa represents the sequencing length and the ordinate represents the number of sequencing. Due to the high error rate of three generations of sequencing, especially the missing errors, the length cannot be accurately measured, so it is a graph similar to a normal distribution.
  • the amplified product is a long DNA fragment of the sense strand tandem antisense strand.
  • the band at about 300bp of the electrophoresis gel corresponds to the sense strand and the antisense strand, self-paired, semi-closed, and single-stranded DNA.
  • the double-stranded DNA strand of the antisense strand is connected to the flattened sense strand corresponding to the band at about 600 bp in the electrophoresis gel.
  • This example is used to illustrate that using a hairpin-like primer to amplify a long DNA fragment of the sense gene tandem antisense strand of the target gene through a one-step RPA reaction.
  • a 276 bp amplicon was amplified from the Lambda DNA standard as a target fragment, the sequence is:
  • a new front primer 5'-CGTAGCTGATGGCACATATGTGCCATCAGCTACGCCACTTCAGCACGAGATGCGGTGGC-3 'and a new back primer 5'-GGCTGCAAGCCCGATAATTATCGGGCTTGCAGCCGCGATGCTGATACCGCACTTCCCGC-3' were used to perform an RPA reaction on the target fragment.
  • RPA reagent was purchased from the British company TwistDX and was named TwistAmp Basic reagent.
  • the reaction system is 50ul, containing TwistAmp Basic powder, 2.4ul pre-amplification primer (10uM), 2.4ul post-amplification primer (10uM), 29.5ul reaction buffer, 1ul target fragment (about 1nM), 2.5ul Magnesium acetate (280 mM), 12.2 ul of water. Reaction conditions: 37 ° C, 40 minutes.
  • the final amplified product was detected by electrophoresis, with obvious bands at about 300bp and 600bp.
  • Three-generation sequencing of the amplified products (ONT instrument).
  • the ligation method was used to detect the amplified products.
  • the abscissa represents the sequencing length, and the ordinate represents the number of sequencing. Due to the high error rate of three generations of sequencing, especially the deletion error, the length cannot be accurately determined, so a 600-bp-like pattern is normal. There is also a small amount of distribution in the longer part of the sequence.
  • the amplified products are mainly long DNA fragments (diploid) of the tandem antisense strand of the sense strand, and there are also a small number of products (tetraploid, 8ploid, etc.) that are repeatedly tandem.
  • the band at about 300bp of the electrophoresis gel corresponds to the sense strand and the antisense strand, which are self-paired, semi-closed, and single-stranded DNA.
  • the double-stranded DNA strand of the antisense strand after the flattened sense strand corresponds to a band at about 600 bp in the electrophoresis gel. Because the amount of amplification products of other lengths is relatively small, it is not clearly shown on the gel map.
  • This embodiment is used to illustrate that using hairpin-shaped primers and adding primers multiple times during the RPA reaction can amplify multiple repeated long DNA fragments of the sense gene tandem antisense strand of the target gene.
  • a 405 bp amplicon was amplified from the Lambda DNA standard as a target fragment, the sequence is:
  • a new pre-primer 5'-TATATATATATATATATATATATATATATATATATATATATAGGGAAGCTTGGAAAGTGTTCGACGGTGAGCTGAG-3 'and a new post-primer 5'-TATATATATATATATATATATATATATATATATAGGGAAGCTTATACTGTCATCAGCATTACGTCATC-3' are used to perform an RPA reaction on the target fragment.
  • RPA reagent was purchased from the British company TwistDX and was named TwistAmp Basic reagent.
  • the reaction system is 50ul, containing TwistAmp Basic powder, 1ul of primers before amplification (10uM), 1ul of primers after amplification (10uM), 29.5ul of reaction buffer, 1ul of target fragment (about 1nM), 2.5ul of acetic acid. Magnesium (280mM), 12.2ul of water. Reaction conditions: 37 ° C, 150 minutes.
  • amplification primer mixture including pre-amplification primer (5uM), post-amplification primer (5uM)
  • amplification primer mixture add 2ul of amplification primer mixture at 20 minutes
  • amplification primer add amplification primer at 40 minutes 2ul of the mixture
  • 2ul of the amplification primer mixture was added at the 60th minute
  • 2ul of the amplification primer mixture was added at the 80th minute.
  • the electrophoresis gel map of the final amplified product has a clear band at about 450bp, 900bp, 1350bp, 1800bp, and a diffuse band at 5000bp to the sample port.
  • Three-generation sequencing of the amplified product using the interrupt method to detect the amplified product.
  • the sequencing structure shows that most of the repeated tandem structures are within 6 times, but the high-fold repeat sequences with a read length of more than 5000bp account for about 5 %.
  • the amplified product is a long DNA fragment with a sense strand and a tandem antisense strand.
  • the tandem repeats are widely distributed and have high-fold repeats.

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Abstract

本发明公开了一种核酸的恒温扩增引物及其应用，所述引物包括用于对目的基因进行扩增的前引物和后引物，所述前引物为在5'端具有外源的自我配对的发夹结构的序列，或在5'端具有与所述目的基因中靠近所述前引物端内的任意源序列相匹配所形成发夹结构的序列。本发明效果为：利用本发明的引物进行核酸扩增技术产生出目的基因的序列重复串联的结构，并且重复的次数可做精细的控制，该技术还可应用在三代测序技术上，扩增步骤不仅简单快速，还可提高测序的准确率。

Description

核酸的恒温扩增引物及其应用 技术领域
本发明涉及到分子生物学技术领域，尤其涉及一种核酸的恒温扩增引物及其应用。
背景技术
核酸扩增技术广泛的用于核酸检测领域，包括核酸序列测定。常规的核酸扩增技术包括PCR(聚合酶链式反应)技术，以及等温扩增技术RPA(重组酶聚合酶扩增)，HAD(依赖解旋酶的核酸恒温扩增技术)等，这些技术能够快速扩增核酸的数量，但不能改变核酸的长度，不能产生首尾串联结构的长链核酸。另外一些核酸扩增技术，比如RCA(滚环扩增)，LAMP(环介导等温扩增法)等等，虽然能够产生长链核酸，但反应不能控制，不能精细的调控串联结构中的重复片段数量。
虽然传统的核酸检测中，只需要扩增核酸的数量，但在一些特殊的场合，产生重复的串联结构却显得非常有用，比如在第三代核酸测序中，由于采用了单分子测序技术，测序的准确度有限，目前无论ONT(牛津纳米孔ONT)，还是PacBio公司的测序仪器对核酸单次测序的准确率都在90％左右，满足不了特需应用。
其中PacBio公司(太平洋生物公司)采用CCS(循环比对测序)的模式，即对单个核酸分子反复多次测序，最终准确率达到99.99％以上。ONT公司对同一DNA双链分别测序后，也就是获得两次重复，经过软件校准，也能显著的提升准确率到96％左右。一些实验室采用了比较特殊的扩增手段，产生了更多次重 复的串联结构，获得了更高的准确率，比如INC-Seq:accurate single molecule reads using nanopore sequencing(DOI 10.1186/s13742-016-0140-7)等等，但这些方法步骤多，时间长，成本高，难以推广。因此，现有的扩增技术，要么不能产生串联结构的长链核酸，要么产生的核酸长度不能精细的控制。
在三代测序技术中，通过对同一序列反复测序可以提高测序准确率，但是需要消耗仪器本身的产出通量，如果反复测两次，最终产出数据就要减少大约一半，如果反复测序10次，产出数据就要减少大约90％。通常反复测两次是性价比最高的模式，据报道，反复测6次后对准确率的提升出现明显边际递减效应。所以如果能够合理的控制扩增片段中重复次数，并且扩增步骤简单，快速，就有比较重要的现实意义。
发明内容
鉴于现有技术存在上述缺陷，本发明的目的在于提供一种核酸的恒温扩增引物及其应用。
本发明的目的，将通过以下技术方案得以实现：
核酸的恒温扩增引物，所述引物包括用于对目的基因进行扩增的前引物和后引物，所述前引物为在5’端具有外源的自我配对的发夹结构的序列，或在5’端具有与所述目的基因中靠近所述前引物端内的任意源序列相匹配所形成发夹结构的序列。
优选地，所述后引物与完成目的基因扩增时所需的下游引物序列相同或所述后引物为在完成目的基因扩增时所需的下游引物序列相同的序列及其在所述下游引物的5’具有外源的自我配对的发夹结构的序列组成的序列。
优选地，应用于可产生对目的基因进行串联重复序列的扩增产物的核酸恒温扩增的方法。
优选地，所述应用方法包括但不限于为RPA扩增方法、HAD扩增方法、PCR扩增方法。
优选地，所述扩增产物应用于对测序仪器的文库制备。
一种应用以上任意所述引物产生串联重复序列的核酸恒温扩增的方法，其特征在于：包括如下步骤，
S1、通过上游引物及下游引物进行核酸扩增形成DNA目的片段，所述目的片段由正义链和反义链构成；
S2、在扩增时加入前引物和后引物进行双引物扩增；其中，所述前引物为在5’端具有外源的自我配对的发夹结构的序列，或在5’端具有与所述目的基因中靠近所述前引物端内的任意源序列相匹配所形成发夹结构的序列；
S3、通过再次添加后引物，进行单引物扩增，形成所需的具有正义链与反义链在同一条链上串联的结构的扩增产物。
优选地，当所述S2中后引物具有与下游引物序列相同序列时，所述S2形成在一端引入可形成发夹环序列的扩增产物，所述S3形成具有正义链与反义链在同一条链上串联一次结构的2倍体扩增产物；
当所述S2中后引物为5’端具有外源的自我配对的发夹结构的序列时，所述S2形成具有正义链与反义链在同一条链上串联一次结构的2倍体扩增产物，所述S3形成具有正义链与反义链在同一条链上串联两次结构的4倍体扩增产物。
优选地，当所述S2中后引物为5’端具有外源的自我配对的发夹结构的序列时，重复N次所述S3中单引物扩增步骤，所述S3形成除了具有以正义链与反义链在同一条链上串联两次结构的4倍体扩增产物为主外，还将形成多种具有以正义链与反义链在同一条链上重复串联多次为辅的多倍体扩增产物，每种所述多倍体之间以指数级上升，且每种所述多倍体数量比将随着重复次数增加而提高，所述N≥1。例如，当重复S3后，产生的产物除了以4倍体的扩增产 物为主，还会伴随着产生8倍体和16倍体为辅的扩增产物；当再次基础上，再进行重复，则产生的8倍体和16倍体的比例将大大提高，同时，还伴有32倍体和64倍体的扩增产物产生，以此类推，其扩增产物的比例受到扩增效率的限制，在此不做明确的限制。
优选地，所述引物产生串联重复序列的核酸恒温扩增的方法，还包括如下步骤，
S4、将S3获得的重复串联的扩增产物看作一个新的整体，在两端引入新的差异序列作为新的引物，分别再依次进行S2双引物扩增及S3单引物扩增步骤的重复，获得两次以上的重复串联结构的扩增产物，所述S4中引入差异序列的方法包括且不限于为通过连接反应接上Y型接头，且该差异序列为S4扩增中的新引物。
本发明突出效果为：1、利用本发明的引物进行核酸扩增技术产生出目的基因的序列重复串联的结构，并且重复的次数可做精细的控制，比如2次，4次，8次等重复。
2、该技术还可应用在三代测序技术上，扩增步骤不仅简单快速，还可提高测序的准确率。比如ONT公司的测序仪器，采用修饰的引物扩增，扩增产物可以直接和测序引物通过化学方法结合，省去了连接反应的步骤，从现有的耗时60分钟缩减至5分钟，大大提高了效率。
以下便结合实施例附图，对本发明的具体实施方式作进一步的详述，以使本发明技术方案更易于理解、掌握。
附图说明
图1：本发明提及的介绍RPA技术中扩增产物的电泳胶图。
图2：利用本发明的引物进行核酸扩增的步骤示意图，其中前引物为具有发夹结构的引物。
图3：利用本发明的引物进行核酸扩增的步骤示意图，其中前、后引物为均具有发夹结构的引物。
图4：扩增出重复串联两次的多倍体为主的扩增产物的步骤示意图。
图5：本发明实施例1的测序读长图。
图6：本发明实施例2的测序读长图。
具体实施方式
本发明揭示了一种核酸的恒温扩增引物及其应用，所述引物包括用于对目的基因进行扩增的前引物和后引物，所述前引物为在5’端具有外源的自我配对的发夹结构的序列，或在5’端具有与所述目的基因中靠近所述前引物端内的任意源序列相匹配所形成发夹结构的序列。所述后引物与完成目的基因扩增时所需的下游引物序列相同或是在完成目的基因扩增时所需的下游引物序列相同的序列及其在所述下游引物的5’具有外源的自我配对的发夹结构的序列组成的序列。核酸的恒温扩增引物可应用于RPA扩增方法、HAD扩增方法、PCR扩增方法以产生对目的基因进行串联重复序列的扩增产物。
为更好的理解本发明，首先，介绍一下RPA技术的特点，传统的PCR方法中，每次循环中通过温度升高到95℃来打开模板DNA双链，温度下降到60℃的过程中，引物结合到模板DNA(脱氧核糖核酸)的匹配部分，在RPA的反应过程中，无须加热解开双链，在常温下比如37℃，重组酶会结合引物在模板DNA双链中扫描，发现互补的匹配序列后，发生置换反应，把引物结合上去，同时具有链置换能力的DNA聚合酶开始合成新的DNA。
在第一篇公开介绍RPA技术的出版物中(PLoS Biol.2006Jul；4(7):e204.DNA detection using recombination proteins)，在文献的补充图表中，即如本发明图1所示，横坐标是目的条带预期的长度，M是参照DNA，纵坐标是参照DNA的条带的长度，在扩增产物的电泳胶图上，作者用*标识了 一些和预期长度不一样的DNA条带，他们是预期长度的2倍和3倍，作者推测了这些产物是由于形成了自身配对的发夹结构，产生了非特异性的扩增产生的。而这些2倍和3倍于目的核酸长度的扩增产物，是传统RPA扩增中要避免的，并且也是微弱和不稳定出现的，但它揭示了RPA扩增有这个潜力。本技术方案基于该基础上，设计了新的引物，通过RPA技术实现了核酸长度增加的扩增产物，当然也可以通过HAD的技术实现，还可以通过其他有链置换能力的DNA聚合酶参与，并且能让引物不断的结合到目的片段上的扩增反应来实现。
1.首先把目的DNA片段扩增形成正义链和反义链串联结构的产物。
结合图2所示，DNA目的片段(Target A+是正义链，Target A-是反义链)是由前引物(Primer F)和后引物(Primer R)扩增出的扩增片段。
步骤一，进行RPA反应的同时加入后引物和新的前引物(Primer LoopA+F),新的前引物3’端的序列是和Primer F一致的，5’端的序列可以形成一个发夹形状的环。步骤一的扩增产物在一端引入了一个可以形成发夹环的序列。
步骤二，进行RPA反应的同时只加入后引物，进行单引物扩增。扩增反应以Target A+为模板，源源不断的产生Target A-。
步骤三，新生成的Target A-，在3’端可以形成发夹环，可以以自身为模板，继续合成，产物是一条自我配对的，半封闭的，DNA单链。
步骤四，后引物继续参与RPA反应，新的扩增产物是5’端是Target A-序列，3’端是Target A+序列的双链，长度约是目的片段的2倍。
通过设计了在目的片段一侧能够形成发夹结构的引物，利用另一侧的引物 进行单引物的RPA的扩增，实现了稳定产生正义链串联反义链的扩增产物。
其中为了形成发夹结构，引物可以在5’端自带一个反向互补的序列，也可以在5’端自带一个序列，此序列可以和扩增子的内部靠近5’端的某一部分序列反向互补。
2.把目的DNA片段扩增形成正义链和反义链多次重复串联结构的产物。
如图2所示，按以上方法能够把目的片段扩增成正义链串联反义链的扩增产物，把此扩增产物看成一个新的目的片段，通过在两侧引入新的类似序列(比如通过连接反应，加上新的接头序列)，就能扩增出原目的片段正义链串联反义链重复两次的扩增产物。同理，重复这样的步骤，可以扩增出重复2的n次方的扩增产物，其中n是任意整数，实际操作中，n受到扩增效率的限制。
3.快速把目的DNA片段扩增形成正义链和反义链串联结构的产物。
为了能够快速扩增出正义链串联反义链的扩增产物，也可以采用都具有发夹结构的扩增前后引物。
如图3所示，由于引物的5’端是一个闭合的环形结构，自身已经配对，导致互补链的3’端的互补结构一旦暴露出来，只能也自身配对，然后以自身为模板进行扩增，形成正义链串联反义链的，半封闭的，DNA单链。同时新的引物延长生成的新链，被置换游离后，3’端也会自身配对，以自身为模板进行扩增，形成正义链串联反义链的，半封闭的，DNA单链。
所以扩增完成后的产物直接就是正义链串联反义链的，半封闭的，DNA单链(2倍体,目的DNA片段的正义链和反义链串联一次形成的新的DNA片段)。由于2倍体产物还能进行扩增，所以也会有更长的扩增产物出现，比如4倍体(目的DNA片段的正义链和反义链重复串联二次形成的新的DNA片段)， 6倍体(目的DNA片段的正义链和反义链重复串联三次形成的新的DNA片段)，8倍体(目的DNA片段的正义链和反义链重复串联四次形成的新的DNA片段)等等，他们都是2的整数倍，但是受到扩增效率的限制，2倍体占绝大多数。如果扩增引物分批多次添加，可以显著增加多倍体的比例。
4.把目的DNA片段扩增形成正义链和反义链两次重复串联结构的产物。
如图4所示，起始片段是图2的扩增产物，主要是正义链串联反义链的2倍体，如果反应结束后，继续添加带发夹结构的扩增后引物，进行单引物扩增，就可以继续扩增出4倍体为主的扩增产物。也可以在图2所示步骤中，直接使用带发夹结构的后引物，扩增完成后，产物以4倍体为主。同理，图3的扩增产物，再次使用带发夹结构的扩增前引物或后引物，进行单引物扩增，也可以继续扩增出重复串联两次的4倍体为主的扩增产物。
5.用HAD实现扩增出串联结构的产物。
本技术方案也可以通过HDA的方法实现，首先介绍一下HDA的技术特点。HDA的扩增过程可以细分为解旋酶解开DNA双链、单链结合蛋白结合并维持单链、引物结合、聚合酶结合、催化靶序列合成、解旋酶解新链再循环6个阶段。在反应开始时，解旋酶在辅助蛋白的辅助下与双链DNA结合，在ATP参与下开始把双链DNA解成单链。出现的单链DNA马上被体系中的单链结合蛋白结合，稳定刚解开的DNA单链，防止重新形成双链。单链模板形成后，引物随即与靶序列结合。同时聚合酶结合并催化合成靶序列。新合成的序列则进人新一轮的解链扩增循环。经过不断的循环，靶序列得到指数式扩增，产物跟常规PCR的产物一样，都是双链靶DNA。
因为HAD反应也能在同一温度下，一边完成DNA链的置换合成反应， 一边完成新的引物结合到模板上引发新的合成反应。所以采用和RPA类似的引物和步骤也可以扩增出正义链串联反义链一次或者多次重复的扩增片段。
但是，由于HAD反应在没有引物的情况下，体系中的解旋酶也能打开DNA双链，所以就会出现双链动态的分离和结合的情况。在3’端具有自身互补的回文序列的时候，在双链分离到重新配对的过程中，有一定的概率形成正义链和反义链完全匹配的情况，也有一定的概率可以在每条链的回文序列处，各自形成一个茎环结构，3’端的茎环结构可以引发新的扩增，以自身为模板，长度再扩增一倍。
如果目的DNA片段两侧都有能形成茎环结构的序列，类似图3的扩增产物，那么在没有引物的情况下，目的片段也能自发的不断变长，扩增出正义链串联反义链多次重复的扩增片段。重复次数和反应时间相关。
所以采用HAD技术进行扩增的时候，如果进行单引物扩增中使用的单引物没有茎环结构，反应可以像RPA反应一样，精确的控制片段的长度，如果扩增引物采用了茎环结构，反应在引物耗尽的情况下，仍然可以增加长度，随着扩增时间的延长，出现更多次的重复。
实施例1：
本实施例用于说明利用发夹状的引物，通过两步RPA反应，扩增出目的基因正义链串联反义链的长DNA片段。
使用前引物：5’-CCACTTCAGCACGAGATGCGGTGGC-3’，和后引物：5’-GCGATGCTGATACCGCACTTCCCGC-3’，从Lambda DNA标准品上PCR扩增出一段276bp的扩增子作为目的片段，序列为：
Figure PCTCN2019091694-appb-000001
Figure PCTCN2019091694-appb-000002
使用新的前引物5’-CGTAGCTGATGGCACATATGTGCCATCAGCTACGCCACTTCAGCACGAGATGCGGTGGC-3’和后引物对目的片段进行RPA反应。RPA试剂购自英国TwistDX公司，名称为TwistAmp Basic试剂。
第一步反应体系是50ul，含TwistAmp Basic干粉，2.4ul的扩增前引物(10uM)，2.4ul的扩增后引物(10uM)，29.5ul的反应缓冲液，1ul的目的片段(约1nM)，2.5ul的醋酸镁(280mM)，12.2ul的水。反应条件：37℃，40分钟。
第二步反应体系是50ul，含TwistAmp Basic干粉，4.8ul的扩增后引物(10uM)，29.5ul的反应缓冲液，1ul的第一步扩增产物100倍稀释液，2.5ul的醋酸镁(280mM)，12.2ul的水。反应条件：37℃，40分钟。
最终扩增产物进行电泳检测，在约300bp和600bp处有明亮条带。
扩增产物三代测序(ONT仪器)，采用连接法，检测扩增产物，长度在600bp处集中。如图5所示，横坐标代表测序长度，纵坐标代表测序数量，由于三代测序的高错误率，尤其是缺失错误，不能精准的测定长度，所以是一个类似正态分布的图形。
结论：1、扩增产物为正义链串联反义链的长DNA片段。2、电泳胶约 300bp处的条带对应的是正义链串联反义链的，自我配对的，半封闭的，DNA单链。电泳胶约600bp处的条带对应的展平后的正义链串联反义链的DNA双链。
实施例2：
本实施例用于说明利用发夹状的引物，通过一步RPA反应，扩增出目的基因正义链串联反义链的长DNA片段。
使用前引物：5’-CCACTTCAGCACGAGATGCGGTGGC-3’，和后引物：5’-GCGATGCTGATACCGCACTTCCCGC-3’，从Lambda DNA标准品上PCR扩增出一段276bp的扩增子作为目的片段，序列为：
Figure PCTCN2019091694-appb-000003
使用新的前引物5’-CGTAGCTGATGGCACATATGTGCCATCAGCTACGCCACTTCAGCACGAGATGCGGTGGC-3’和新的后引物5’-GGCTGCAAGCCCGATAATTATCGGGCTTGCAGCCGCGATGCTGATACCGCACTTCCCGC-3’对目的片段进行RPA反应。RPA试剂购自英国TwistDX公司，名称为TwistAmp Basic试剂。
反应体系是50ul，含TwistAmp Basic干粉，2.4ul的扩增前引物(10uM)， 2.4ul的扩增后引物(10uM)，29.5ul的反应缓冲液，1ul的目的片段(约1nM)，2.5ul的醋酸镁(280mM)，12.2ul的水。反应条件：37℃，40分钟。
最终扩增产物进行电泳检测，在约300bp和600bp处有明显条带。
扩增产物三代测序(ONT仪器)，采用连接法，检测扩增产物，长度在600bp处有高峰。如图6所示，横坐标代表测序长度，纵坐标代表测序数量，由于三代测序的高错误率，尤其是缺失错误，不能精准的测定长度，所以在600bp处是一个类似正态分布的图形，在序列更长的部分也有少量分布。
结论：1、扩增产物主要为正义链串联反义链的长DNA片段(2倍体)，也有少量多次重复串联的产物(4倍体，8倍体等)。2、电泳胶约300bp处的条带对应的是正义链串联反义链的，自我配对的，半封闭的，DNA单链。电泳胶约600bp处的条带对应的展平后的正义链串联反义链的DNA双链，其他长度的扩增产物由于量比较少，在胶图上没有明显的显示。
实施例3：
本实施例用于说明利用发夹状的引物，在RPA反应过程中多次添加引物，可以扩增出目的基因正义链串联反义链的多次重复的长DNA片段。
使用前引物：5’-GGAAAGTGTTCGACGGTGAGCTGAG-3’，和后引物：5’-ATACTGTCATCAGCATTACGTCATC-3’，从Lambda DNA标准品上PCR扩增出一段405bp的扩增子作为目的片段，序列为：
Figure PCTCN2019091694-appb-000004
Figure PCTCN2019091694-appb-000005
使用新的前引物5’-TATATATATATATATATATATATATATATAGGGAAGCTTGGAAAGTGTTCGACGGTGAGCTGAG-3’和新的后引物5’-TATATATATATATATATATATATATATATAGGGAAGCTTATACTGTCATCAGCATTACGTCATC-3’对目的片段进行RPA反应。RPA试剂购自英国TwistDX公司，名称为TwistAmp Basic试剂。
反应体系是50ul，含TwistAmp Basic干粉，1ul的扩增前引物(10uM)，1ul的扩增后引物(10uM)，29.5ul的反应缓冲液，1ul的目的片段(约1nM)，2.5ul的醋酸镁(280mM)，12.2ul的水。反应条件：37℃，150分钟。
在反应过程中，添加扩增引物混合物(含扩增前引物(5uM)，扩增后引物(5uM))，在第20分钟时候添加扩增引物混合物2ul，在第40分钟时候添加扩增引物混合物2ul，在第60分钟时候添加扩增引物混合物2ul，在第80分钟时候添加扩增引物混合物2ul。
最终扩增产物电泳胶图，在约450bp处，在900bp处，在1350bp处，在1800bp处有清晰条带，在5000bp到加样口处有弥散条带。
扩增产物三代测序(ONT仪器)，采用打断法，检测扩增产物，测序结构显示绝大部分是6次以内的重复串联结构，但是读长在5000bp以上的高倍数 重复序列，约占5％。
结论：扩增产物为正义链串联反义链的长DNA片段，串联的重复次数分布比较广，而且有高倍数重复序列。
本发明尚有多种实施方式，凡采用等同变换或者等效变换而形成的所有技术方案，均落在本发明的保护范围之内。

Claims (9)

  1. 核酸的恒温扩增引物，其特征在于：所述引物包括用于对目的基因进行扩增的前引物和后引物，所述前引物为在5’端具有外源的自我配对的发夹结构的序列，或在5’端具有与所述目的基因中靠近所述前引物端内的任意源序列相匹配所形成发夹结构的序列。
  2. 根据权利要求1所述的核酸的恒温扩增引物，其特征在于：所述后引物与完成目的基因扩增时所需的下游引物序列相同或所述后引物为在完成目的基因扩增时所需的下游引物序列相同的序列及其在所述下游引物的5’具有外源的自我配对的发夹结构的序列组成的序列。
  3. 如权利要求1所述的核酸的恒温扩增引物的应用，其特征在于：应用于可产生对目的基因进行串联重复序列的扩增产物的核酸恒温扩增的方法。
  4. 如权利要求3所述的核酸的恒温扩增引物的应用，其特征在于：所述应用方法包括但不限于为RPA扩增方法、HAD扩增方法、PCR扩增方法。
  5. 如权利要求3所述的核酸的恒温扩增引物的应用，其特征在于：所述扩增产物应用于对测序仪器的文库制备。
  6. 一种应用如权利要求1或2中任意所述引物产生串联重复序列的核酸恒温扩增的方法，其特征在于：包括如下步骤，
    S1、通过上游引物及下游引物进行核酸扩增形成DNA目的片段，所述目的片段由正义链和反义链构成；
    S2、在扩增时加入前引物和后引物进行双引物扩增；其中，所述前引物为在5’端具有外源的自我配对的发夹结构的序列，或在5’端具有与所述目的基因中靠近所述前引物端内的任意源序列相匹配所形成发夹结构的序列；
    S3、通过再次添加后引物，进行单引物扩增，形成所需的具有正义链与反义链在同一条链上串联的结构的扩增产物。
  7. 一种应用如权利要求6中任意所述引物产生串联重复序列的核酸恒温 扩增的方法，其特征在于：当所述S2中后引物具有与下游引物序列相同序列时，所述S2形成在一端引入可形成发夹环序列的扩增产物，所述S3形成具有正义链与反义链在同一条链上串联一次结构的2倍体扩增产物；
    当所述S2中后引物为5’端具有外源的自我配对的发夹结构的序列时，所述S2形成具有正义链与反义链在同一条链上串联一次结构的2倍体扩增产物，所述S3形成具有正义链与反义链在同一条链上串联两次结构的4倍体扩增产物。
  8. 一种应用如权利要求6所述引物产生串联重复序列的核酸恒温扩增的方法，其特征在于：当所述S2中后引物为5’端具有外源的自我配对的发夹结构的序列时，重复N次所述S3中单引物扩增步骤，所述S3形成除了具有以正义链与反义链在同一条链上串联两次结构的4倍体扩增产物为主外，还将形成多种具有以正义链与反义链在同一条链上重复串联多次为辅的多倍体扩增产物，每种所述多倍体之间以指数级上升，且每种所述多倍体数量比将随着重复次数增加而提高，所述N≥1。
  9. 一种应用如权利要求7所述引物产生串联重复序列的核酸恒温扩增的方法，其特征在于：还包括如下步骤，
    S4、将S3获得的重复串联的扩增产物看作一个新的整体，在两端引入新的差异序列作为新的引物，分别再依次进行S2双引物扩增及S3单引物扩增步骤的重复，获得两次以上的重复串联结构的扩增产物，所述S4中引入差异序列的方法包括且不限于为通过连接反应接上Y型接头，且该差异序列为S4扩增中的新引物。
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