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Method used for synthesis of DNA library with fixed length and specific terminal sequence based on template material

A DNA library, terminal sequence technology, applied in the field of synthetic biology

Active Publication Date: 2019-08-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the current technical status, the present invention can convert naturally occurring DNA or nucleic acid into an sgRNA library. This method does not require a reference sequence to design sgRNA, but directly converts natural DNA into DNA of a fixed length, such as 19-24bp, these Fixed-length fragments are derived from DNA material, so there is no inaccurate reference sequence design

Method used

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  • Method used for synthesis of DNA library with fixed length and specific terminal sequence based on template material
  • Method used for synthesis of DNA library with fixed length and specific terminal sequence based on template material
  • Method used for synthesis of DNA library with fixed length and specific terminal sequence based on template material

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0083] 1. Example 1 Starting material: Mouse embryonic stem cell H3K4me3 chromatin immunoprecipitation DNA

Embodiment 2

[0084] 2. Example 2 Starting material: Human embryonic stem cell H3K4me3 chromatin immunoprecipitation DNA

[0085] 3.NEBNext ultra-fast end repair / add dA tail module, NEB, E7442

[0086] 4. NEBNext ultra-fast connection module, NEB, E7445;

[0087] 5. Ampure XP beads, Beckman, A63881;

[0088] 6.Dynabeads TM T1 magnetic beads, Invitech, 65602;

[0089] 7.2X binding and washing buffer;

[0090] 8. Nuclease-free water, Ambion,10977023;

[0091] 9.1M sodium hydroxide, Sigma, S2770-100ML;

[0092] 10. Hybridization buffer;

[0093] 11. Extension buffer;

[0094] 12.3' hydroxyl recovery buffer;

[0095] 13. Washing buffer 1;

[0096] 14. Washing buffer 2;

[0097] 15.10X MBN buffer, NEB, M0250;

[0098] 16.MBN (10U / μL), NEB, M0250;

[0099] 17.10X T4DNA ligation buffer, NEB, B0202;

[0100] 18. T4 polynucleotide kinase, NEB, M0201;

[0101] 19. T4 DNA ligase, NEB, M0202;

[0102] 20. Qiaquick glue recovery kit, Qiagen, 28704;

[0103] 21. Qiaquick PCR product recovery kit, Qiagen, 28104;

[0104] 22.10X...

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Abstract

The invention belongs to the field of biological field, and more specifically relates to a method used for synthesis of a DNA library with a fixed length and a specific terminal sequence based on a template material. The method comprises following steps: fragmentation double chain DNA obtaining; DNA terminal restoration and 3' terminal tailing; 3'Biotin modified A1 joint connection; connection product purification; fixing of DNA onto a streptavidin containing solid phase; denaturation of double chain DNA into single chain DNA; primer 1 hybridization; reversible chemical modification of dNTPs extension basic group with deoxyribosyl 3' terminal hydroxyl; pentose 3' terminal hydroxyl restoration; extension of DNA of fixed length; 5' terminal trimming; tail end sequence selection; tail end sequence removing; and connection of joint used for molecular cloning. The method is capable of realizing construction of the library with region fixed length, specific terminal sequence, and high sequence variability, without reference genome, and library construction cost is reduced greatly.

Description

Technical field [0001] The invention relates to the field of synthetic biology, in particular to a method for preparing a DNA library with a fixed length and a specific end sequence based on a template material. Background technique [0002] There are a set of regulatory molecular biological mechanisms behind the life process and biological behavior. The laws of these molecular mechanisms are the central law, that is, the law of information transmission between DNA, RNA, and protein. In the past 30 years, the methods for studying biological mechanisms have mainly been forward genetics and reverse genetics. Forward genetics is based on phenomena to find key molecules and mechanisms. Reverse genetics is based on molecules and reveals through manipulation of molecules. Mechanism and find the corresponding biological process. For forward genetics research, one of the major technical methods is genetic screening. In the early stage, unbiased screening is used for cellular mechanisms....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/10
CPCC12N15/1068C40B50/06C12Q2521/501C12Q2521/301
Inventor 贾俊岭潘辰李然
Owner ZHEJIANG UNIV
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