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Method for introducing site-specific mutagenesis rapidly and efficiently

A site-directed mutagenesis and high-efficiency technology, applied in the field of genetic engineering, can solve the problems of complicated operation, high cost, low transformation efficiency, etc., and achieve the effect of strong verifiability and strong operability.

Inactive Publication Date: 2014-02-19
TIANJIN UNIV OF SCI & TECH
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, to a certain extent, there are problems of cumbersome operation, low conversion efficiency and high cost.

Method used

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  • Method for introducing site-specific mutagenesis rapidly and efficiently
  • Method for introducing site-specific mutagenesis rapidly and efficiently
  • Method for introducing site-specific mutagenesis rapidly and efficiently

Examples

Experimental program
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Embodiment 1

[0022] Example 1: A method for introducing point mutations quickly and efficiently

[0023] Previous experiments showed that yeast strain fil1 (gifted by Professor Johan M. Thevelein of the Belgian Institute of Plant Microbiology) has obvious high temperature tolerance, and this excellent trait is caused by the mutation of the 1682 amino acid at the C-terminal of the CYRI gene of the yeast. In this example, the site-directed mutation at this position was introduced into the industrial yeast BY14a (purchased from the China Industrial Microbial Culture Collection Management Center, Address: Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, 100015, preservation number CICC31616, Saccharomyces cerevisiae).

[0024] Yeast genome extraction kit and plasmid extraction kit were purchased from Solarbio, and the restriction endonucleases and competent cells DH5α used were purchased from Takara.

[0025] The composition of the complete medium without uracil in this examp...

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Abstract

The invention discloses a method for introducing site-specific mutagenesis rapidly and efficiently. The method provided by the invention can realize the introduction of site-specific mutagenesis by using an integrated plasmid YIplac211 as the carrier, selecting URA3 gene carried by the integrated plasmid as selection marker, and carrying out homologous recombination of direct repeats twice. According to the invention, no exogenous gene remains during the introduction of site-specific mutagenesis so as to ensure the safety of strains. Additionally, the method provided by the invention can also use other industrial strains besides yeast.

Description

Technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for introducing site-directed mutations quickly and efficiently. Background technique [0002] With the penetration of molecular biology technology into various fields of biology, biologists pay more and more attention to the function research of genes, purposely changing the bases encoding specific amino acids, obtaining mutant proteins, and studying the relationship between protein structure and function. The site-directed mutagenesis technology has emerged as an important means of genetic engineering operations and is widely used in the medical field. At present, there are mainly three methods commonly used in site-directed mutagenesis: (1) oligonucleotide primer-mediated mutation, (2) PCR-mediated mutation, such as overlap extension PCR, large primer method, etc., (3) cassette type mutation. Based on the principle of the above method, kits for site-directed mutagenesi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 肖冬光刘广新董建张翠英
Owner TIANJIN UNIV OF SCI & TECH
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