Gene site-directed multi-site mutation method

Abstract

The invention relates to a gene site-directed multi-site mutation method, which comprises the following steps of: respectively designing a forward mutation primer and a reverse mutation primer according to sites of sequences where mutated bases are located, wherein the mutation sites are positioned in the middle of the forward primer and the position close to 5' end of the reverse primer; designing the same pairs of mutation primers according to the number of the mutation sites; taking a plasmid containing methylated sites as a template, carrying out the first round of PCR reaction on the first pair of primers with high fidelity polymerase for amplifying a gene fragment containing the first mutation base; adopting DpnI enzyme for carrying out the restriction enzyme digestion on the gene fragment, removing the template plasmid, obtaining a ring plasmid with a target mutation site and an opening; then taking the ring plasmid as the template and sequentially adding the residual mutation primers for PCR reaction for obtaining a ring plasmid with a plurality of mutation sites and an opening. The method utilizes a complementary region of the primers to complete the polymerase chain reaction of the ring plasmid template with the opening, thereby introducing the site-directed mutation with more than one site.

Description

【Technical field】 [0001] The invention relates to the field of genetic engineering, in particular to a method for gene-directed multi-site mutation. 【Background technique】 [0002] With the completion of the Human Genome Project, more and more biological workers have shifted their research focus to gene function. Gene site-directed mutagenesis technology is meeting this requirement. Purposely mutating one or several amino acids on the protein chain to other amino acids can determine the structure and function center of the protein; mutating one or several bases of the cis-responsive element on the promoter can explore the function of the element function center. In addition, site-directed mutagenesis technology also provides a way for genetic engineering, optimization, and improvement of enzyme activity. [0003] Many companies in the market now have kits for single-point mutations, such as: foreign ones include invitrogen, clontech, and stratagene; domestic ones include ...

AI Technical Summary

Problems solved by technology

However, their method requires phosphorylation of the 5' end of the primer, annealing alone, extension with T4 DNA polymerase and ligation with T4 ligase, and finally PCR, which is slightly expensive and cumbersome to operate

In 2005, Danish Jensen et al. published a method for short-distance polygenic mutations, which was a supplement to stratagene's multi-site directed mutagenesis kit, which made up for the inability of the kit to perform short-distance polygenic mutations. defect

However, this method requires two steps of gel recovery in addition to PCR, which is slightly complicated

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