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Method for precisely editing genome DNA sequence

A DNA sequence and genome technology, applied in the field of precise genome DNA sequence editing, can solve problems such as inability to delete, and achieve the effect of increasing the incidence rate

Inactive Publication Date: 2016-05-11
WUHAN DANMIYOU BIOLOGICAL MEDICAL
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  • Summary
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Problems solved by technology

Because the principles of these two technologies are completely different, and based on the existing theoretical knowledge of DNA repair, even if the two are combined, only a specific sequence can be inserted, but a specific sequence cannot be deleted.

Method used

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  • Method for precisely editing genome DNA sequence
  • Method for precisely editing genome DNA sequence
  • Method for precisely editing genome DNA sequence

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Experimental program
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Embodiment Construction

[0056] 1. Experimental materials and methods:

[0057] 1.1 Experimental materials

[0058] CD4 + U87 cells (gifted by Academician Zeng Yi of Beijing University of Technology), commonly used restriction enzymes and T4 DNA ligase were purchased from NEB (Beijing) Co., Ltd. TaqDNA high-fidelity polymerase was purchased from Life Technology; PBS, trypsin digestion solution, double antibody, TaqPCR Mastermix, PCR product purification kit, plasmid mini-prep kit, and DNA molecular weight standards were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Cell culture flasks, pipettes, centrifuge tubes, etc. were purchased from Corning Company. FBS was purchased from Gibco, and DMEM and DMEM / F12 were purchased from HyClone. SDS-PAGE gel rapid preparation kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Opti-MEM optimized culture medium and liposome 2000 were purchased from Invitrogen, and G418 and puromycin were purchased from Shanghai Sangon...

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Abstract

The invention provides a method for precisely editing a genome DNA sequence. A TALEN, CRISPR / Cas9 and other gene targeting technologies and homologous recombination technologies are utilized in a combined mode for precisely editing the genome DNA sequence. According to the method, the technical problem that in the prior art, a specific sequence cannot be deleted is solved, the specific base sequence can be seamlessly inserted or deleted in a fixed point mode, and seamless free modification of the genome DNA sequence of eukaryocyte is achieved for the first time.

Description

Technical field: [0001] The invention relates to a method for precise editing of genome DNA sequences. Specifically, it involves the combined use of gene targeting technology and traditional homologous recombination technology to insert or delete specific base sequences in the genome in a fixed-point and seamless manner. Background technique: [0002] In the field of genetic engineering, researchers have been trying to modify the base sequence of the genome in order to inactivate a gene and obtain the function of a new gene. [0003] Escherichia coli homologous recombination is a new genetic engineering technology developed in the late 1990s. It can precisely complete genetic modification such as fragment insertion, deletion and sequence change without relying on restriction enzyme sites. Escherichia coli homologous recombination technology is often used for targeted modification of chromosomal DNA - gene knock-in and knock-out, and gap-repair (gap-repair) method to obtain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
CPCC12N15/85C12N2800/107C12N2810/10
Inventor 李东升丁妍卢智勇
Owner WUHAN DANMIYOU BIOLOGICAL MEDICAL
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