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WO2025076733A1 - Procédé de construction rapide d'une banque d'acides nucléiques - Google Patents

Procédé de construction rapide d'une banque d'acides nucléiques Download PDF

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Publication number
WO2025076733A1
WO2025076733A1 PCT/CN2023/124061 CN2023124061W WO2025076733A1 WO 2025076733 A1 WO2025076733 A1 WO 2025076733A1 CN 2023124061 W CN2023124061 W CN 2023124061W WO 2025076733 A1 WO2025076733 A1 WO 2025076733A1
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WO
WIPO (PCT)
Prior art keywords
linker
dna
buffer
dna polymerase
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2023/124061
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English (en)
Chinese (zh)
Inventor
夏军
孔格致
杨林
张艳艳
刘萍
王逸丛
赵霞
陈芳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MGI Tech Co Ltd
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MGI Tech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MGI Tech Co Ltd filed Critical MGI Tech Co Ltd
Priority to PCT/CN2023/124061 priority Critical patent/WO2025076733A1/fr
Publication of WO2025076733A1 publication Critical patent/WO2025076733A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • 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
    • 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 fifth aspect of the present invention aims to provide a nucleic acid library.
  • Ligase which can catalyze the intermolecular or molecular ligation of single-stranded oligonucleotides (single-stranded RNA and/or single-stranded DNA), or single nucleotides. Forming a phosphodiester bond between the 5'-P terminus and the 3'-OH terminus within the ribosome; and
  • the linker comprises a first linker and a second linker.
  • the first connector also includes a sample tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, such as a barcode sequence or an index sequence.
  • a sample tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, such as a barcode sequence or an index sequence.
  • the first adapter further comprises a unique molecular identifier (UMI) sequence for counting the copy number of the nucleic acid molecule in the sample.
  • UMI unique molecular identifier
  • the second adapter further comprises a unique molecular identifier (UMI) sequence for counting the copy number of the nucleic acid molecule in the sample.
  • UMI unique molecular identifier
  • the length of the unique molecular tag is 5 to 20 bp.
  • the end repair enzyme comprises a polymerase.
  • the end repair enzyme further comprises polynucleotide kinase.
  • the polymerase comprises at least one of T4 DNA polymerase, DNA polymerase I (Klenow) large fragment (Klenow fragment, Klenow Fragment), T7 DNA polymerase, and DNA polymerase I; and further comprises at least one of T4 DNA polymerase and DNA polymerase I (Klenow) large fragment (Klenow fragment, Klenow Fragment).
  • the ligase comprises at least one of T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA/RNA circular ligase (ssDNA/RNA CircLigase), and truncated version of T4 RNA ligase 2; and further comprises: T4 RNA ligase 1.
  • the reagent combination is used for DNA end repair-adapter ligation of DNA fragments.
  • the DNA fragments include at least one of naturally occurring small molecule DNA (such as cfDNA, DNA in formalin-fixed paraffin-embedded samples), DNA fragments obtained by amplification (such as MDA products, cDNA products, amplicons, etc.), and DNA fragments obtained by shearing (the shearing method can be a chemical shearing method (such as enzymatic cutting) or a physical shearing method (such as ultrasonic shearing method and mechanical shearing method)).
  • naturally occurring small molecule DNA such as cfDNA, DNA in formalin-fixed paraffin-embedded samples
  • DNA fragments obtained by amplification such as MDA products, cDNA products, amplicons, etc.
  • shearing method can be a chemical shearing method (such as enzymatic cutting) or a physical shearing method (such as ultrasonic shearing method and mechanical shearing method)).
  • the breaking enzyme comprises a nuclease; further comprises a non-specific nuclease; further comprises at least one of nuclease DNase I, micrococcal nuclease (MNase), nuclease dsDNase, salt-active nuclease SAN, and nuclease Vvn; and further comprises nuclease DNase I.
  • MNase micrococcal nuclease
  • nuclease dsDNase nuclease dsDNase
  • salt-active nuclease SAN salt-active nuclease SAN
  • nuclease Vvn nuclease DNase I.
  • the third aspect of the present invention provides a library construction kit, comprising the reagent combination of the first aspect of the present invention or the reagent combination of the second aspect of the present invention.
  • the kit further comprises: a DNB preparation reagent.
  • the DNB preparation reagent comprises: polynucleotide kinase.
  • the concentration of the polymerase in the end repair enzyme in the system is 0.01 U/ ⁇ L to 1 U/ ⁇ L.
  • the concentration of the ligase in the system is 0.1 U/ ⁇ L to 10 U/ ⁇ L.
  • the concentration of the nucleic acid elongase in the system is 0.005 U/ ⁇ L to 1 U/ ⁇ L.
  • the DNA fragment is the DNA fragment in the first aspect of the present invention.
  • the reaction procedure comprises: reaction at 16-40° C. for 10-60 min; reaction at 50-75° C. for 5-60 min.
  • the concentration of the DNA in the system is 1 nM to 100 nM.
  • the DNA is the DNA in the second aspect of the present invention.
  • the reaction procedure comprises: reaction at 16-40°C for 10-60 min; reaction at 50-75°C for 5-60 min; further comprises: reaction at 37°C for 15 min; reaction at 65°C for 5-10 min.
  • linear amplification is performed first and then rolling circle amplification.
  • the rolling circle amplification and cyclization reaction adopt the DNB preparation reagent in the third aspect of the present invention, that is, the cyclization reaction and rolling circle amplification can be completed in one step or in multiple steps.
  • the DNA fragment is the DNA fragment in the first aspect of the present invention.
  • the concentration of calcium ions in the buffer in the reaction system is 0.001-50 mM.
  • FIG1 is a schematic diagram of the process of rapidly constructing a nucleic acid library according to the present invention.
  • FIG. 2 is a schematic diagram of a single-chain connector preferably used in the present invention.
  • End repair enzymes which are used to remove the 3' overhangs of the DNA fragments and/or fill in the 5' overhangs of the DNA fragments to ultimately form double-stranded DNA fragments with blunt ends (i.e., end repair reactions);
  • a ligase which can catalyze the formation of a phosphodiester bond between the 5'-P end and the 3'-OH end of a single-stranded oligonucleotide (single-stranded RNA and/or single-stranded DNA) or a single nucleotide, or between molecules or within a molecule, and is used for connecting the end-repaired DNA fragment to a linker (i.e., a linker ligation reaction); and
  • Nucleic acid extender is used to extend the complementary chain of the linker connecting the DNA fragments (extending the complementary chain with the linker as a template, i.e., linker extension reaction), thereby obtaining a DNA fragment with double-stranded linkers at both ends.
  • the other nucleotides other than the nucleotide at the 3' end of the linker are independently selected from ribonucleotides (rNTP) and deoxyribonucleotides (dNTP); that is, the other nucleotides other than the nucleotide at the 3' end of the linker can be ribonucleotides (rNTP) or deoxyribonucleotides (dNTP), as shown in Figure 2.
  • rNTP ribonucleotides
  • dNTP deoxyribonucleotides
  • the first linker comprises a linker sequence.
  • the first adapter further comprises a sequencing primer sequence.
  • the first adapter also includes a sample tag sequence for distinguishing different samples to facilitate subsequent mixed sequencing of multiple samples. It can be a barcode sequence or an index sequence.
  • the unique molecular index (UMI) sequence of the second adapter is the same as or different from the unique molecular index (UMI) sequence of the first adapter.
  • the length of the unique molecular tag is 5 to 20 bp.
  • the DNA fragments can be subjected to DNA end repair-adapter ligation without undergoing interruption treatment (those skilled in the art can determine whether interruption treatment is required based on common knowledge and/or conventional technical means).
  • sequence of the connector is as shown in SEQ ID NO.1, 2.
  • the system further comprises the above-mentioned buffer.
  • the concentration of dNTP in the buffer in the system is 0.05-5 mM.
  • the concentration of the linker in the system is 100 nM to 10 ⁇ M; further 200 nM to 800 nM.
  • the concentration of the polymerase in the terminal repair enzyme in the system is 0.01 U/ ⁇ L to 1 U/ ⁇ L; further 0.0725 U/ ⁇ L to 0.29 U/ ⁇ L.
  • the concentration of the ligase in the system is 0.1 U/ ⁇ L to 10 U/ ⁇ L; further 0.1 U/ ⁇ L to 0.4 U/ ⁇ L.
  • the reaction procedure comprises: reacting at 16-40°C for 10-60 min, so that the end repair enzyme can remove the 3' protruding end of the DNA fragment and/or fill the 5' protruding end of the DNA fragment to finally form a double-stranded DNA fragment with a flat end (it can also include dephosphorylating the 3' end of the DNA fragment and phosphorylating the 5' end) (i.e., an end repair reaction occurs), and the ligase can connect the end-repaired DNA fragment with the adapter (i.e., adapter ligation reaction); reacting at 50-75°C for 5-60 min, so that the nucleic acid extension enzyme can extend the complementary chain of the adapter connected to the DNA fragment (extending the complementary chain with the adapter as a template, i.e., adapter extension reaction), thereby obtaining a DNA fragment with double-stranded adapters at both ends, and at the same time, inactivating the end repair enzyme and the ligase.
  • the end repair enzyme can remove the 3' pro
  • a shearing enzyme to break DNA (such as genomic DNA) into DNA fragment
  • the reagent combination is used to perform DNA shearing-end repair-adapter ligation in the same reaction system; wherein the same reaction system is specifically performed in the same reaction container.
  • the concentration of dNTP in the buffer in the system is 0.05-5 mM.
  • the concentration of the linker in the system is 100 nM to 10 ⁇ M; further 200 nM to 800 nM.
  • the concentration of the polynucleotide kinase in the terminal repair enzyme in the system is 0.01 U/ ⁇ L to 1 U/ ⁇ L.
  • the concentration of the ligase in the system is 0.1 U/ ⁇ L to 10 U/ ⁇ L; further 0.1 U/ ⁇ L to 0.4 U/ ⁇ L.
  • the kit further comprises: a DNB preparation reagent for preparing the linear library into DNA nanoballs (DNBs).
  • DNBs DNA nanoballs
  • the DNB preparation reagent is a substance selected from any one of the following DNB preparation methods: a1) single-chain cyclization (cyclization reaction), linear single-chain digestion, purification, and rolling circle replication (rolling circle amplification) are performed in sequence to obtain DNA nanoballs; a2) single-chain cyclization and rolling circle replication are completed in one step to obtain DNA nanoballs; further preferably, the DNB preparation reagent is a substance selected from the following DNB preparation methods: a2) single-chain cyclization and rolling circle replication are completed in one step to obtain DNA nanoballs.
  • the DNB preparation reagent comprises: polynucleotide kinase (preferably T4 polynucleotide kinase), which is used to phosphorylate the 5' end of the linker to facilitate the circularization reaction.
  • polynucleotide kinase preferably T4 polynucleotide kinase
  • a method for constructing a nucleic acid library comprising the steps of using the reagent combination of the first aspect of the present invention, further specifically as follows: DNA fragments, linkers, end repair enzymes, ligases and nucleic acid extension enzymes are mixed, and the resulting system is reacted.
  • a method for DNA shearing-end repair-connector ligation comprising the steps of using the reagent combination of the second aspect of the present invention, further specifically as follows: DNA, a connector, a shearing enzyme, an end repair enzyme, a ligase and a nucleic acid extension enzyme are mixed, and the resulting system is reacted.
  • a method for constructing a nucleic acid library comprising the steps of using the reagent combination of the second aspect of the present invention, further specifically as follows: DNA, linkers, breaking enzymes, end repair enzymes, ligases and nucleic acid extension enzymes are mixed, and the resulting system is reacted.
  • the 3' overhang of the DNA fragment is cut off and/or the 5' overhang of the DNA fragment is filled by the end repair enzyme to finally form a double-stranded DNA fragment with a flat end (it may also include 3' end dephosphorylation and 5' end phosphorylation of the DNA fragment), the end-repaired DNA fragment is connected to the adapter by the ligase, and the complementary chain of the adapter connected to the DNA fragment is extended by the nucleic acid extension enzyme (the complementary chain is extended using the adapter as a template), so as to obtain a DNA fragment with double-stranded adapters at both ends, and then DNA end repair and adapter connection are achieved in one reaction system; compared with the existing DNA end repair-adapter connection method in enzyme digestion library construction, the end A addition step is reduced, and at the same time, DNA end repair and adapter connection can be achieved in one reaction system, and the DNA fragment is directly reacted in one step and then the adapter is added to realize one-step library construction, which can greatly reduce
  • the concentration of the buffer in the system is 10-100 mM.
  • the concentration of magnesium ions and/or manganese ions in the buffer in the system is 1-50 mM.
  • the concentration of dNTP in the buffer in the system is 0.05-5 mM.
  • the concentration of ATP and/or GTP in the buffer in the system is 0.1-10 mM.
  • the concentration of the polynucleotide kinase in the terminal repair enzyme in the system is 0.01 U/ ⁇ L to 1 U/ ⁇ L.
  • the concentration of the nucleic acid elongase in the system is 0.005 U/ ⁇ L to 1 U/ ⁇ L; further 0.005 U/ ⁇ L to 0.02 U/ ⁇ L.
  • the concentration of the DNA fragment in the system is 1 nM to 100 nM.
  • the DNA fragment is the DNA fragment in the first aspect of the present invention.
  • the DNA is the DNA in the second aspect of the present invention.
  • the amplification reaction is at least one of linear amplification and rolling circle amplification; further, rolling circle amplification (ie, the nucleic acid library construction method is a PCR-free nucleic acid library construction method).
  • the rolling circle amplification further comprises a circularization reaction.
  • the cyclization reaction further comprises a phosphorylation reaction (preferably a 5' end phosphorylation reaction).
  • a phosphorylation reaction preferably a 5' end phosphorylation reaction.
  • the phosphorylation reaction uses polynucleotide kinase (preferably T4 polynucleotide kinase).
  • polynucleotide kinase preferably T4 polynucleotide kinase
  • the concentration of the buffer in the reaction system is 10-100 mM.
  • the concentration of dNTP in the buffer in the reaction system is 0.05-5 mM.
  • the concentration of ATP and/or GTP in the buffer in the system is 0.1-10 mM.
  • the concentration of the linker in the reaction system is 100 nM to 10 ⁇ M; further 200 nM to 800 nM.
  • the concentration of the ligase in the reaction system is 0.1 U/ ⁇ L to 10 U/ ⁇ L; further 0.1 U/ ⁇ L to 0.4 U/ ⁇ L.
  • the concentration of the nucleic acid elongase in the reaction system is 0.005 U/ ⁇ L to 1 U/ ⁇ L; further 0.005 U/ ⁇ L to 0.02 U/ ⁇ L.
  • the seventh aspect of the present invention provides a sequencing method, comprising the following steps: obtaining a nucleic acid library; sequencing; the nucleic acid library is the nucleic acid library of the fifth aspect of the present invention.
  • the sequencing further includes the following steps: library quality inspection.

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Abstract

Procédé de construction rapide d'une banque d'acides nucléiques, relevant du domaine technique de la construction de banques. L'invention porte sur une combinaison de réactifs pour la ligature de lieurs pour la réparation des extrémités de l'ADN. Une enzyme de réparation des extrémités excise un surplomb en 3' d'un fragment d'ADN et/ou comble un surplomb en 5' du fragment d'ADN pour former finalement un fragment d'ADN double brin à extrémités émoussées ; une ligase ligature le fragment d'ADN à extrémités réparées à l'aide de lieurs ; une enzyme d'extension de l'acide nucléique étend les brins complémentaires des lieurs ligaturant le fragment d'ADN, de manière à obtenir un fragment d'ADN avec des lieurs double brin aux deux extrémités, ce qui permet d'effectuer la réparation d'extrémité et la ligature de lieurs dans un seul système de réaction et de réduire les étapes de la technique du « A-tailing » et de purification. L'ajout des lieurs à l'ADN dans une réaction en une seule étape permet la construction d'une banque en une seule étape.
PCT/CN2023/124061 2023-10-11 2023-10-11 Procédé de construction rapide d'une banque d'acides nucléiques Pending WO2025076733A1 (fr)

Priority Applications (1)

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PCT/CN2023/124061 WO2025076733A1 (fr) 2023-10-11 2023-10-11 Procédé de construction rapide d'une banque d'acides nucléiques

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101967476A (zh) * 2010-09-21 2011-02-09 深圳华大基因科技有限公司 一种基于接头连接的DNA PCR-Free标签文库构建方法
WO2013104106A1 (fr) * 2012-01-10 2013-07-18 北京贝瑞和康生物技术有限公司 Procédé de construction d'une banque de séquençage de l'adn plasmatique et nécessaire à cet effet
CN106661631A (zh) * 2014-06-06 2017-05-10 康奈尔大学 使用组合的核酸酶、连接酶、聚合酶和测序反应识别和枚举核酸序列、表达、拷贝或dna甲基化变化的方法
US20180142235A1 (en) * 2012-01-10 2018-05-24 Jianguang Zhang Method for constructing a plasma dna sequencing library
WO2021048720A1 (fr) * 2019-09-09 2021-03-18 Immagina Biotechnology S.R.L. Procédé de préparation d'un échantillon d'arn pour séquençage et kit associé
CN112708619A (zh) * 2020-12-30 2021-04-27 纳昂达(南京)生物科技有限公司 Mgi平台的建库用接头、试剂盒及建库方法
CN113249457A (zh) * 2021-06-21 2021-08-13 南京实践医学检验有限公司 一种一步法构建dna纳米球的试剂盒和方法
CN114807305A (zh) * 2022-04-13 2022-07-29 首都医科大学附属北京口腔医院 一种构建原核生物单细胞rna测序文库的方法
CN115715323A (zh) * 2020-06-19 2023-02-24 深圳华大智造科技股份有限公司 一种高兼容性的PCR-free建库和测序方法
WO2023141829A1 (fr) * 2022-01-26 2023-08-03 深圳华大智造科技股份有限公司 Procédé pour effectuer simultanément un séquençage d'adn de génome entier et une méthylation d'adn de génome entier et/ou un séquençage d'hydroxyméthylation

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101967476A (zh) * 2010-09-21 2011-02-09 深圳华大基因科技有限公司 一种基于接头连接的DNA PCR-Free标签文库构建方法
WO2013104106A1 (fr) * 2012-01-10 2013-07-18 北京贝瑞和康生物技术有限公司 Procédé de construction d'une banque de séquençage de l'adn plasmatique et nécessaire à cet effet
US20180142235A1 (en) * 2012-01-10 2018-05-24 Jianguang Zhang Method for constructing a plasma dna sequencing library
CN106661631A (zh) * 2014-06-06 2017-05-10 康奈尔大学 使用组合的核酸酶、连接酶、聚合酶和测序反应识别和枚举核酸序列、表达、拷贝或dna甲基化变化的方法
WO2021048720A1 (fr) * 2019-09-09 2021-03-18 Immagina Biotechnology S.R.L. Procédé de préparation d'un échantillon d'arn pour séquençage et kit associé
CN115715323A (zh) * 2020-06-19 2023-02-24 深圳华大智造科技股份有限公司 一种高兼容性的PCR-free建库和测序方法
CN112708619A (zh) * 2020-12-30 2021-04-27 纳昂达(南京)生物科技有限公司 Mgi平台的建库用接头、试剂盒及建库方法
CN113249457A (zh) * 2021-06-21 2021-08-13 南京实践医学检验有限公司 一种一步法构建dna纳米球的试剂盒和方法
WO2023141829A1 (fr) * 2022-01-26 2023-08-03 深圳华大智造科技股份有限公司 Procédé pour effectuer simultanément un séquençage d'adn de génome entier et une méthylation d'adn de génome entier et/ou un séquençage d'hydroxyméthylation
CN114807305A (zh) * 2022-04-13 2022-07-29 首都医科大学附属北京口腔医院 一种构建原核生物单细胞rna测序文库的方法

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