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WO2024138550A1 - Procédé d'amplification d'acide nucléique et son utilisation dans le séquençage - Google Patents

Procédé d'amplification d'acide nucléique et son utilisation dans le séquençage Download PDF

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Publication number
WO2024138550A1
WO2024138550A1 PCT/CN2022/143516 CN2022143516W WO2024138550A1 WO 2024138550 A1 WO2024138550 A1 WO 2024138550A1 CN 2022143516 W CN2022143516 W CN 2022143516W WO 2024138550 A1 WO2024138550 A1 WO 2024138550A1
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nucleic acid
concatemer
copy
dna
stranded dna
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English (en)
Chinese (zh)
Inventor
夏军
杨林
孔格致
张艳艳
陈芳
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to PCT/CN2022/143516 priority Critical patent/WO2024138550A1/fr
Priority to CN202280102856.2A priority patent/CN120435569A/zh
Publication of WO2024138550A1 publication Critical patent/WO2024138550A1/fr
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • 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 present disclosure relates to the field of molecular biology technology, specifically, the present disclosure relates to the field of nucleic acid amplification, and more specifically, the present disclosure relates to a nucleic acid amplification method, a single-stranded DNA multi-copy concatemer and uses thereof.
  • sequencing technology has become one of the most commonly used and important research methods. Since Sanger invented the dideoxy chain termination method, the first generation sequencing technology, in 1977, and the promotion of the Human Genome Project, the first generation sequencing technology has greatly improved the speed and throughput of sequencing from the initial isotope labeling plus plate electrophoresis to fluorescent labeling and capillary electrophoresis and automated imaging systems. However, compared with the growing demand for sequencing, the developed first generation sequencing technology still cannot meet the needs. Therefore, the next generation sequencing technology was invented and developed rapidly in a short period of time. The next generation sequencing technology is also called the second generation sequencing technology or massively parallel sequencing technology.
  • the conventional steps of large-scale parallel sequencing technology are to fragment DNA, add adapters with known sequences to form a so-called library, and then load these DNA fragments with adapters onto the sequencing chip.
  • Each DNA fragment with adapters in the library occupies a certain position on the chip, so that all the sequences on the chip are sequenced at one time, thereby achieving the purpose of large-scale parallel sequencing.
  • an extremely important step is what we call the sequencing template signal amplification step.
  • the technologies for amplifying sequencing DNA templates to form so-called base signal acquisition units can be mainly divided into three types: microbead emulsion PCR amplification, solid phase bridge PCR amplification, and DNA nanoball amplification.
  • Microbead emulsion PCR amplification is mainly used in Roche's 454 sequencing platform and Thermo Fisher's SOLiD and Ion torrent platforms.
  • the specific steps are to mix the DNA library with the adapter sequence with the microbeads with complementary sequences to the adapter, dNTP, primers and DNA polymerase and other reactants, add specific mineral oil and surfactant, and then use an oscillator to oscillate violently to make the reaction system form a stable water-in-oil emulsion.
  • each droplet will contain only one magnetic bead and a single-stranded DNA.
  • at least 10 to the power of 6 ideal droplets can be formed in 1mL of emulsion.
  • the surface of each bead is covered with thousands of copies of the same DNA sequence. Subsequently, the emulsion mixture is broken, and the amplified fragments are still bound to the magnetic beads.
  • Solid-phase bridge PCR amplification is mainly used in various Illumina sequencing platforms.
  • fragmented DNA is connected to adapters to form a library, and the library with adapters is converted into single strands, which are then diluted to a suitable concentration and added to the sequencing chip.
  • the single-stranded DNA fragments are bound to the sequencing chip through complementary adapters. Because the concentration of the library is low enough after dilution, it can be considered that the library fragments are evenly bound to the chip surface, and the positions where each fragment is bound are far enough apart.
  • the single-stranded DNA hybridized with the complementary adapter on the chip is amplified to form a double strand, and the unfixed complementary single strand is washed away, leaving the amplified chain, whose free end can hybridize with other nearby adapter primers to form a bridge structure.
  • it is denatured into a single strand and interacts with other nearby adapter primers again to form a bridge, becoming the template for the next round of amplification and continuing amplification.
  • each single copy of the DNA molecule is amplified nearly a thousand times to become a monoclonal DNA cluster.
  • Microbead emulsion PCR amplification The copy number of the DNA sequencing template is high and can be amplified to thousands of copies. However, in order to ensure the copy number, the volume of the microbeads is relatively large, and the diameter is generally in the micrometer range, ranging from 1 micron to tens of microns, which affects the sequencing throughput. The use of small microbeads will increase the number of microbeads contained in a droplet, affecting the accuracy of sequencing. In addition, corresponding instruments are required when preparing droplets, which is costly and complicated to operate.
  • DNA nanoball amplification Linear amplification makes the number of DNA template copies in the DNA nanoball not high, generally a few hundred copies.
  • the structure of the DNA nanoball is loose, and increasing the number of copies will greatly affect the volume of the DNA nanoball, affecting the improvement of sequencing throughput and sequencing length.
  • increasing the number of copies or long-fragment DNA templates requires high continuous synthesis ability of the chain displacement polymerase.
  • the structure of the DNA nanoball is loose, and before it is loaded onto the chip, its structure is easily destroyed, resulting in the need for extra caution in its operation, which increases the complexity of the operation.
  • the present disclosure aims to solve one of the technical problems in the related art at least to some extent.
  • nucleic acid amplification method comprises:
  • nucleic acid amplification method comprises:
  • the nucleic acid amplification method disclosed in the present invention is easy to operate and does not require complex instruments.
  • the obtained single-stranded DNA multiple copies are small in size, large in number of copies, and stable in structure, which effectively solves the problem that the DNA nanoballs obtained by the rolling circle amplification method are difficult to operate, loose in structure, large in size, and low in amplification efficiency. If it is applied to the field of sequencing, it can save the chip usage area, reduce costs, and further improve the accuracy of sequencing due to its large copy number.
  • any one primer in the primer pair contains restriction site 1, the other primer does not contain restriction site, or contains restriction site 2 different from restriction site 1;
  • the enzyme cleavage site 1 is cleaved by enzyme 1, and the enzyme cleavage site 2 is cleaved by enzyme 2, and enzyme 1 and enzyme 2 are different.
  • enzyme 1 is used to cut the restriction site 1 contained in the double-stranded DNA multi-copy concatemer to form a single-stranded gap in the double-stranded DNA loop, and the strand with the gap is digested with a digestive enzyme to obtain a first single-stranded DNA multi-copy concatemer.
  • At least one primer in the primer pair carries a modified ribonucleotide or deoxyribonucleotide, and the restriction site is cut by the enzyme to form a gap on any one of the circular DNAs in the double-stranded DNA concatemer.
  • the nucleic acid amplification method further comprises, after step (D):
  • the amplification and ligation reaction system contains a high temperature resistant DNA ligase.
  • the amplification and ligation reaction system contains a high-temperature-intolerant DNA ligase.
  • the amplification and ligation reaction system when the amplification and ligation reaction system contains a thermolabile DNA ligase, it further includes a DNA helicase and a single-stranded binding protein.
  • the single-stranded binding protein contained in the system can prevent the unwound DNA from self-pairing.
  • the enzyme 1 and the enzyme 2 include enzymes capable of cleaving the ribonucleotides and/or uracil deoxyribonucleotides.
  • the digestive enzyme comprises an enzyme capable of digesting linear DNA.
  • the digestive enzyme comprises a DNA exonuclease.
  • the DNA exonuclease includes at least one selected from DNA exonuclease I, DNA exonuclease III, T5 Exonuclease, Exonuclease T, Exonuclease VII, and Lambda Exonuclease.
  • Another aspect of the present disclosure provides a method for increasing the copy number of a sequencing template. According to an embodiment of the present disclosure, the method comprises:
  • the aforementioned nucleic acid amplification method is used to obtain multiple single-stranded DNA multi-copy concatemers to achieve multi-copy amplification of the sequencing template.
  • the above-mentioned nucleic acid amplification method is used to perform multi-copy amplification on the sequencing template, and the obtained single-stranded DNA multi-copy concatemer is loaded onto the sequencing chip as the base signal acquisition unit in sequencing, which has the following advantages: the DNA sequencing template of the DNA multi-copy concatemer has a large number of copies and a small volume, thereby occupying a small area of the sequencing chip or flow cell; the increase in the number of copies of the DNA sequencing template will not affect its volume, and the fragment length of the DNA sequencing template has little effect on the volume of the DNA multi-copy concatemer, which is conducive to improving the sequencing read length.
  • the structure of the DNA multi-copy concatemer is stable, convenient for operation and long-term storage, and has low requirements for the DNA polymerase used in the reaction.
  • Another aspect of the present disclosure provides a single-stranded DNA multi-copy concatemer.
  • the single-stranded DNA multi-copy concatemer is prepared by the aforementioned nucleic acid amplification method.
  • the single-stranded DNA circular molecule further comprises a tag sequence, and the tag sequence is located between the two known nucleic acid sequences.
  • the single-stranded DNA multi-copy concatemer further comprises a plurality of linear DNA single strands, the 5' end of each linear DNA single strand being an amplification primer capable of hybridizing to a known sequence and a nucleic acid sequence complementary to the nucleic acid fragment to be detected.
  • the 5' end of the linear DNA single strand is free, and the 3' end is hybridized on the concatemer.
  • the present disclosure provides a nucleic acid composite molecule, which includes the above-mentioned single-stranded DNA multi-copy concatemer and multiple linear DNA single strands, and the 5' end of the linear DNA single strand is an amplification primer that can hybridize to a known sequence and a nucleic acid sequence complementary to the nucleic acid fragment to be detected.
  • Another aspect of the present disclosure provides uses of the single-stranded DNA multi-copy concatemer prepared by the aforementioned nucleic acid amplification method, the aforementioned single-stranded DNA multi-copy concatemer, and the aforementioned nucleic acid composite molecule in sequencing.
  • the present disclosure provides a method for producing a second strand, the method comprising using the aforementioned single-stranded DNA multi-copy concatemer as a template, hybridizing with a primer that is fully or partially complementary to a known nucleic acid sequence, and performing a rolling circle amplification reaction under the action of a polymerase to generate a second strand.
  • the primer is free in the solution.
  • the aforementioned single-stranded DNA multi-copy concatemer can be used as a base signal acquisition unit and serially loaded on a sequencing chip.
  • the single-stranded DNA multi-copy concatemer is small in size, has a large number of copies, and a stable structure, which effectively solves the problems of difficult operation, loose structure, large size, and low amplification efficiency of DNA nanoballs obtained by rolling circle amplification.
  • the aforementioned single-stranded DNA multi-copy concatemer is applied to the sequencing field, saving chip usage area, reducing costs, and improving accuracy.
  • Another aspect of the present disclosure provides a single-stranded DNA multi-copy concatemer prepared by the aforementioned nucleic acid amplification method, and uses of the aforementioned single-stranded DNA multi-copy concatemer in template amplification and micro-amplification.
  • FIG1 shows a schematic flow chart of a nucleic acid amplification method according to an embodiment of the present disclosure
  • FIG2 shows a schematic flow chart of a nucleic acid amplification method according to another embodiment of the present disclosure
  • FIG3 shows a technical roadmap of a nucleic acid amplification method according to an embodiment of the present disclosure
  • FIG8 is a schematic diagram showing a comparison of the theoretical sizes of DNA multi-copy concatemers and DNA nanoballs obtained from templates of the same length according to an embodiment of the present disclosure.
  • base also known as nucleobase, nitrogenous base
  • natural bases include adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U); non-natural bases include locked nucleic acids (LNA) and bridged nucleic acids (BNA); base analogs include hypoxanthine, deazaadenine, deazaguanine, deazahypoxanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine.
  • the base type can be used to represent the nucleotide type in the present disclosure.
  • the 5' ends of the primers in the primer pair are all phosphorylated.
  • the 5' ends of the primers in the primer pair are all phosphorylated means that the 5' end of each primer in the primer pair is phosphorylated, which facilitates the ligase to connect the 3' end of the chain amplification product to the 5' end of the primer to form a ring.
  • the multi-copy concatemer of DNA obtained in step (c) or (D) is used as a template, and a primer complementary to the universal sequence of the first single-stranded DNA multi-copy concatemer linker is used to amplify using the Phi29 enzyme. Relying on the chain displacement ability of the Phi29 enzyme, after one circle of amplification on the single-stranded loop, a second single-stranded DNA multi-copy concatemer complementary to the multi-copy concatemer is displaced.
  • the enzyme 1 and enzyme 2 respectively include at least one selected from USER enzyme, RNase H, UDG, RNase HII, and hSMUG1, and enzyme 1 and enzyme 2 are different enzymes.
  • the high temperature resistant DNA ligase includes at least one selected from Taq ligase and AMP ligase.
  • the digestive enzyme comprises an enzyme capable of digesting single-stranded DNA.
  • the method for nucleic acid amplification ( FIG. 4 ) comprises:
  • thermostable ligase such as T4 DNA ligase.
  • T4 DNA ligase an isothermal amplification system is used, which may include DNA helicase, single-stranded binding protein, etc.
  • the primer is free in the solution.
  • a primer 1 complementary to the adapter sequence is synthesized, and the 5' end of primer 1 is paired with the adapter sequence using the principle of base complementarity, and a sequencing-by-synthesis reaction is subsequently performed.
  • the second strand can be obtained by the following method:
  • the adapter as a primer binding site, and the Phi29 enzyme for amplification, relying on the chain displacement ability of the Phi29 enzyme, after amplifying one circle on the single-stranded loop, a second chain complementary to the multi-copy concatemer is displaced.
  • Amplification is performed with primer 2, and a ligation reaction is performed after one circle of amplification to generate a two-strand DNA multi-copy concatemer. Then the USER enzyme is used to cut the one chain to form a gap, and then the DNA digestion enzyme is used to remove the entire one-strand DNA multi-copy concatemer. Finally, a two-strand DNA multi-copy concatemer in a single-stranded state is obtained.
  • the primer 1 and the primer 2 can have different restriction sites in any order, and in addition to U base and ribonucleic acid base, they can also be 5,6-dihydroxythymine, 5-hydroxyuracil, 5-hydroxymethyluracil, and 5-formyluracil, etc.
  • the enzyme 1 and the enzyme 2 include at least one selected from USER enzyme, RNase H, UDG, RNase HII, and hSMUG1, and the enzyme 1 and the enzyme 2 are different enzymes.
  • the single-stranded DNA multi-copy concatemer obtained by the above-mentioned nucleic acid amplification method is loaded onto a sequencing chip as a base signal acquisition unit in sequencing, and has the following advantages: the DNA sequencing template of the DNA multi-copy concatemer has a large number of copies and a small volume, thereby occupying a small area of the sequencing chip or the flow tank; the increase in the number of copies of the DNA sequencing template will not affect its volume, and the fragment length of the DNA sequencing template has a small effect on the volume of the DNA multi-copy concatemer, which is conducive to improving the sequencing read length.
  • the structure of the DNA multi-copy concatemer is stable, convenient for operation and long-term storage, and has low requirements for the DNA polymerase used in the reaction.
  • the DNA multi-copy concatemer does not need to react on the sequencing chip or the flow tank, has low requirements for the chip, and does not need to generate nanowells on the chip and pre-plant oligonucleotides on the chip.
  • the above-mentioned nucleic acid amplification method is not only applicable to the field of sequencing, but also to any field of micro-amplification. When there are few experimental materials and the samples are scarce, the disclosed method can be used to efficiently amplify the sample nucleic acid content without changing the nucleic acid abundance.
  • Primer2 5’P-GCTCACAGAA/rC//rG//rA//rC/ATGGCTACGATCCGAC-3’ (SEQ ID NO: 2)
  • r represents the modification of ribonucleotide and P represents phosphorylation modification.
  • Escherichia coli genomic DNA was used as material, the nucleic acid amplification method disclosed in the present invention was adopted, the obtained amplification product was loaded onto a chip for sequencing, and the results were directly quality checked, which was in line with expectations.
  • the whole process from obtaining the nucleic acid template to obtaining the amplified product is simple to operate, and the required instruments and consumables are also conveniently available to experimental personnel in the field, which improves the experimental efficiency and reduces the experimental cost.
  • Table 1 a large amount of DNC can be obtained with the investment of a small amount of DNA library, and it can be seen from Figure 6 that the quality of the obtained DNC meets the standard, so this method can efficiently amplify the nucleic acid template while ensuring the quality of the amplified product.
  • Figures 7 and 8 that in sequencing, the method disclosed in the present invention can significantly reduce the chip occupied area. Compared with the prior art, under the chip of the same area, the sequencing read length can be effectively improved, and because the template amplification is not performed on the chip, the chip requirements are relatively low.
  • a primer pool for amplifying the tumor hotspot region of the EGFR gene which contains 8 pairs of specific oligonucleotide primers (EGFR_1F-EGFR_8F) and (EGFR_1R-EGFR_8R), with an amplicon size of 100 to 200 bp.
  • the sequences of the specific oligonucleotide primers are shown in Table 6 below.

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Abstract

Sont prévus un procédé d'amplification d'acide nucléique, un concatémère d'ADN multicopie monobrin et l'utilisation de celui-ci. Le procédé consiste à : lier un acide nucléique avec une séquence connue pour obtenir un acide nucléique à amplifier, et effectuer une réaction de ligature d'amplification en utilisant une paire d'amorces conçue sur la base de la séquence connue contenue dans l'acide nucléique à amplifier, ce qui permet d'obtenir un premier concatémère d'ADN multicopie double brin, les deux amorces de la paire d'amorces pouvant comprendre différents sites de clivage enzymatique 1 et 2 respectivement ; utiliser une enzyme 1 pour cliver le site de clivage enzymatique 1 contenu dans le concatémère d'ADN multicopie double brin de façon à former un espace monobrin dans la boucle d'ADN double brin, et utiliser une enzyme digestive pour digérer l'espace monobrin, de façon à obtenir un premier concatémère d'ADN multicopie monobrin ; effectuer une réaction de ligature d'amplification en utilisant une amorce avec le premier concatémère d'ADN multicopie monobrin servant de matrice de façon à obtenir un second concatémère d'ADN multicopie double brin, utiliser une enzyme 2 pour cliver le site de clivage enzymatique 2 contenu dans le second concatémère d'ADN multicopie double brin de façon à former un espace monobrin dans le second concatémère d'ADN multicopie double brin, et utiliser l'enzyme digestive pour digérer l'espace monobrin de façon à obtenir un second concatémère d'ADN multicopie monobrin ; et utiliser le concatémère d'ADN multicopie monobrin mentionné ci-dessus pour le séquençage d'acide nucléique.
PCT/CN2022/143516 2022-12-29 2022-12-29 Procédé d'amplification d'acide nucléique et son utilisation dans le séquençage Ceased WO2024138550A1 (fr)

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PCT/CN2022/143516 WO2024138550A1 (fr) 2022-12-29 2022-12-29 Procédé d'amplification d'acide nucléique et son utilisation dans le séquençage
CN202280102856.2A CN120435569A (zh) 2022-12-29 2022-12-29 一种核酸扩增方法及其在测序中的用途

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101213311A (zh) * 2005-04-29 2008-07-02 J·克雷格·文特尔研究院 利用滚环扩增扩增和克隆单个dna分子
WO2018214036A1 (fr) * 2017-05-23 2018-11-29 深圳华大基因股份有限公司 Méthode d'enrichissement pour région génomique cible sur la base d'une amplification par cercle roulant et son application
CN112534063A (zh) * 2018-05-22 2021-03-19 安序源有限公司 用于核酸测序的方法、系统和组合物
CN112795620A (zh) * 2019-11-13 2021-05-14 深圳华大基因股份有限公司 双链核酸环化方法、甲基化测序文库构建方法和试剂盒
CN113667716A (zh) * 2021-08-27 2021-11-19 北京医院 基于滚环扩增的测序文库构建方法及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101213311A (zh) * 2005-04-29 2008-07-02 J·克雷格·文特尔研究院 利用滚环扩增扩增和克隆单个dna分子
WO2018214036A1 (fr) * 2017-05-23 2018-11-29 深圳华大基因股份有限公司 Méthode d'enrichissement pour région génomique cible sur la base d'une amplification par cercle roulant et son application
CN112534063A (zh) * 2018-05-22 2021-03-19 安序源有限公司 用于核酸测序的方法、系统和组合物
CN112795620A (zh) * 2019-11-13 2021-05-14 深圳华大基因股份有限公司 双链核酸环化方法、甲基化测序文库构建方法和试剂盒
CN113667716A (zh) * 2021-08-27 2021-11-19 北京医院 基于滚环扩增的测序文库构建方法及其应用

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