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Method for quickly constructing gene targeting vector based on site specificity recombination

A carrier and site technology, applied in the fields of biotechnology and genetic engineering, can solve the problems of low homologous recombination efficiency and specificity that need further research, and achieve the effect of rapid construction and simple construction method

Inactive Publication Date: 2011-02-09
FUDAN UNIV
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Problems solved by technology

After the recombination is completed, no unnecessary sequences will be left on the vector, but on the one hand, homologous recombination may produce unexpected chain exchange, and the specificity needs to be further studied; on the other hand, the efficiency of homologous recombination is generally relatively low

Method used

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  • Method for quickly constructing gene targeting vector based on site specificity recombination
  • Method for quickly constructing gene targeting vector based on site specificity recombination
  • Method for quickly constructing gene targeting vector based on site specificity recombination

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Embodiment Construction

[0036] 1. Acquisition of compatible substrate pairs for φBT1 integrase and determination of recombination efficiency.

[0037] (1) Due to the construction of a series of plasmids for detecting the reaction efficiency of mutant substrates: primers primer 1 and primer 2 are complementary, primer 3 and primer 4 are complementary, primer 5 and primer 6 are complementary, primer 7 and primer 8 are complementary; dissolve the above primers to The final concentration is 20 μM. Take 10 μl each and mix with corresponding complementary primers, then cool and anneal in boiling water. After annealing, double-stranded DNA molecules containing wild-type and mutation sites are obtained, and both ends are respectively digested with EcoRI and HindIII. after the same end. Digest with EcoRI and HindIII (system: appropriate amount of plasmid, 5 μl of NEB “10×buffer”, 5 μl of 10×BSA, 1 μl of restriction endonucleases, ddH 2 (2) Supplement the total volume to 50 μl) plasmid pBC-SK (-), and the dig...

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Abstract

The invention belongs to the technical fields of biotechnology and gene engineering, and in particular relates to a method for quickly constructing a gene targeting vector based on phi BT1 integrase and a mutation recognition site thereof. The invention provides three pairs of mutative intergrase recognition sequences by strict mutation screening and verification, and the mutative substrate pair has equal reaction efficiency to that of a wild type, and appears as incompatible in the same system. On the basis, the method constructs a set of related vectors comprising a backbone vector capable of realizing Escherichia coli-streptomyces coelicolor shuttle, and three TA clone vectors containing mutative substrate pairs for constructing and screening homology arms, and provides a concrete method of series recombination and reaction of four DNA fragments in vitro. The method of the invention is simple, quick and high-efficiency, can be used for constructing the gene targeting vector of streptomyces, and can be widely used for quickly constructing gene targeting vectors of different species by corresponding original substitution.

Description

technical field [0001] The invention belongs to the technical fields of biotechnology and genetic engineering, and in particular relates to a method for rapidly constructing a gene targeting vector based on φBT1 integrase and its mutation recognition site. Background technique [0002] Gene targeting technology usually refers to the homologous recombination of DNA fragments containing known sequences and homologous sequences in the genome of recipient cells, integrating into the genome of recipient cells, adding foreign DNA fragments or genes at target sites, or targeting A technique to knock out or increase certain related genes. As an important means of modern molecular biology and genetic manipulation, gene targeting technology plays a vital role in the in-depth analysis and qualitative research of individual genes, genomes and gene families. Through this technology, the directional modification of biological genetic information can be realized, and the modified genetic ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/63C12N15/11C12Q1/02C12N15/09C12N15/66
Inventor 丁晓明张霖张渤赵国屏
Owner FUDAN UNIV
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