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WO2018006567A1 - Procédé hautement efficace pour lier un lieur d'adn - Google Patents

Procédé hautement efficace pour lier un lieur d'adn Download PDF

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Publication number
WO2018006567A1
WO2018006567A1 PCT/CN2016/113506 CN2016113506W WO2018006567A1 WO 2018006567 A1 WO2018006567 A1 WO 2018006567A1 CN 2016113506 W CN2016113506 W CN 2016113506W WO 2018006567 A1 WO2018006567 A1 WO 2018006567A1
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WO
WIPO (PCT)
Prior art keywords
dna
linker
adapter
double
overhang
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.)
Ceased
Application number
PCT/CN2016/113506
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English (en)
Chinese (zh)
Inventor
耿亮
辛文
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.)
BeiJing TransGen Biotech Co Ltd
Original Assignee
BeiJing TransGen Biotech Co Ltd
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Filing date
Publication date
Application filed by BeiJing TransGen Biotech Co Ltd filed Critical BeiJing TransGen Biotech Co Ltd
Publication of WO2018006567A1 publication Critical patent/WO2018006567A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR

Definitions

  • Standard second-generation sequencing library construction involves the following steps: (i) fragmentation, (ii) end-repair, (iii) 5' end phosphorylation, (iv) 3' end plus dA, which can be ligated to the sequencing linker, (v The adaptor is ligated (vi) by PCR to enrich the product of the linker successfully connected at both ends.
  • Figure 6 is a diagram showing the electrophoresis pattern of the ligation reaction in Example 2.
  • Lane M 100bp DNA Ladder
  • Lane 4 120 bp fragment plus A product + S-L Adapter Mix ligation product.
  • the materials and sources used in the examples are: DNA Polymerase, FastPfu PCR SuperMix (-dye), PCR Purification Kit, T4 DNA Ligase (Beijing Quanjin Biotechnology Co., Ltd.), ⁇ DNA (Takara), primer synthesis, sequencing (Life technologies).
  • the annealing product conditions were: 95 ° C for 5 minutes, and slowly cooled to room temperature. 1 ⁇ l was detected by electrophoresis on a 2.0% agarose gel.
  • the reaction conditions were: 25 ° C, 2 hours.
  • Example 2 of the present invention shows that a mixture of three terminal structure linkers is used, and its DNA fragment attachment efficiency is much higher than that of a conventional 3'dT overhang structure, and both ends of the fragment are The success rate of connection to the connector is significantly improved.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur un procédé hautement efficace pour lier un lieur, le procédé consistant à : utiliser une ADN polymérase de type A ou un fragment de Klenow d'ADN polymérase pour traiter une molécule d'ADN de manière à ajouter une extrémité saillante 3'dA ; puis lier la molécule d'ADN traitée à un mélange de lieurs afin d'obtenir un produit de liaison, le mélange de lieurs comprenant trois types de lieurs d'ADN double brin et les extrémités des trois lieurs d'ADN double brin étant respectivement une extrémité franche, une extrémité saillante 3'dT et une extrémité saillante 3'dC. Le procédé peut efficacement améliorer l'efficacité de liaison d'un lieur à l'ADN.
PCT/CN2016/113506 2016-07-08 2016-12-30 Procédé hautement efficace pour lier un lieur d'adn Ceased WO2018006567A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610537963.4 2016-07-08
CN201610537963.4A CN105950612B (zh) 2016-07-08 2016-07-08 一种高效的dna接头连接方法

Publications (1)

Publication Number Publication Date
WO2018006567A1 true WO2018006567A1 (fr) 2018-01-11

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Family Applications (1)

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PCT/CN2016/113506 Ceased WO2018006567A1 (fr) 2016-07-08 2016-12-30 Procédé hautement efficace pour lier un lieur d'adn

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CN (1) CN105950612B (fr)
WO (1) WO2018006567A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950612B (zh) * 2016-07-08 2019-06-21 北京全式金生物技术有限公司 一种高效的dna接头连接方法
CN108048915A (zh) * 2017-12-01 2018-05-18 北京科迅生物技术有限公司 用于ctDNA文库构建的接头混合物、包括其的试剂盒及应用
CN110129415B (zh) * 2019-05-17 2023-08-18 迈杰转化医学研究(苏州)有限公司 一种ngs建库分子接头及其制备方法和用途
CN110565174B (zh) * 2019-09-17 2022-09-30 北京博昊云天科技有限公司 一种dna文库构建方法
CN111041070B (zh) * 2019-12-27 2022-08-19 北京优迅医学检验实验室有限公司 一种高通量测序文库构建的dna转化效率的检测方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102061335A (zh) * 2010-11-15 2011-05-18 苏州众信生物技术有限公司 一种二代高通量测序的不对称dna双链接头及其应用
WO2012013932A1 (fr) * 2010-07-29 2012-02-02 University Court Of The University Of St Andrews Procédé race perfectionné
WO2015103339A1 (fr) * 2013-12-30 2015-07-09 Atreca, Inc. Analyse d'acides nucléiques associés à des cellules individuelles à l'aide de codes-barres d'acides nucléiques
CN104789553A (zh) * 2015-04-08 2015-07-22 浙江圣庭生物科技有限公司 一种血液游离dna文库的构建方法
CN105950612A (zh) * 2016-07-08 2016-09-21 北京全式金生物技术有限公司 一种高效的dna接头连接方法

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WO2012012037A1 (fr) * 2010-07-19 2012-01-26 New England Biolabs, Inc. Adaptateurs oligonucléotidiques : compositions et procédés d'utilisation
CN105400776B (zh) * 2014-09-12 2019-12-31 深圳华大智造科技有限公司 寡核苷酸接头及其在构建核酸测序单链环状文库中的应用
CN105200530A (zh) * 2015-10-13 2015-12-30 北京百迈客生物科技有限公司 一种适用于高通量全基因组测序的多样品混合文库的构建方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012013932A1 (fr) * 2010-07-29 2012-02-02 University Court Of The University Of St Andrews Procédé race perfectionné
CN102061335A (zh) * 2010-11-15 2011-05-18 苏州众信生物技术有限公司 一种二代高通量测序的不对称dna双链接头及其应用
WO2015103339A1 (fr) * 2013-12-30 2015-07-09 Atreca, Inc. Analyse d'acides nucléiques associés à des cellules individuelles à l'aide de codes-barres d'acides nucléiques
CN104789553A (zh) * 2015-04-08 2015-07-22 浙江圣庭生物科技有限公司 一种血液游离dna文库的构建方法
CN105950612A (zh) * 2016-07-08 2016-09-21 北京全式金生物技术有限公司 一种高效的dna接头连接方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAGNUSON, V.L. ET AL.: "Substrate Nucleotide-determined Non-templated Addition of Adenine by Tag DNA Polymerase: Implications for PCR-based Genotyping and Cloning", BIOTECHNIQUES, vol. 21, no. 4, 31 October 1996 (1996-10-31), pages 700 - 709, XP008062075, ISSN: 0736-6205 *

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CN105950612A (zh) 2016-09-21
CN105950612B (zh) 2019-06-21

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