Gene site-directed mutation method based on seamless cloning, and applications thereof

Abstract

The invention relates to a gene site-directed mutation method based on seamless cloning, a kit, and applications of the gene site-directed mutation method based on seamless cloning. The gene site-directed mutation method based on seamless cloning comprises following steps: 5' terminal forward and reverse mutation primers containing complementary homologous arm sequences are designed based on the sequences of mutation sites, wherein the mutation sites may be at any places of homologous arms; 5' terminal forward and reverse mutation primers containing another complementary homologous arm are designed respectively at plasmid non-mutation site zones, PCR amplification is adopted to obtain two segments, wherein one end of each segment contains mutation sites, and more than 500bp difference is formed; a mutation primer with the same pair number of that of sites is designed based on mutation site number; homologous recombinase is adopted for one-step seamless splicing of 2 to 5 segments, andconstruction of an annular plasmide with target mutation sites is realized. Compared with classic overlap extension PCR, the gene site-directed mutation method possesses following advantages: the genesite-directed mutation method is rapid and simple, can be used for single or multiple site, continuous or discontinuous mutation of any sites of a plasmid smaller than 40kb, and mutation efficiency is 100%.

Description

technical field [0001] The invention relates to a gene site-directed mutation method based on seamless cloning technology, a gene site-directed mutation kit prepared based on the method and a specific embodiment of its application, belonging to the technical field of genetic engineering. Background technique [0002] Site-directed mutagenesis based on PCR technology is an essential tool for studying gene regulation, protein structure and function in molecular biology and protein engineering. Gene site-directed mutation methods mainly include base substitution, deletion, insertion, multi-site mutation, and random mutation of one or more sites. Many PCR-based gene site-directed mutagenesis methods have been commercialized, and the most commonly used methods include the following: [0003] (1) Overlap extension PCR method (overlap extension method). The forward and reverse primers used in this method contain complementary overlapping regions. The product amplified by the ove...

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Problems solved by technology

[0007] (1) When the concentration of the PCR amplification product is low, the construction efficiency of the mutant plasmid is very low;

[0008] (2) The high-fidelity DNA polymerase has a weak strand displacement reaction activity, which may trigger the strand displacement reaction and amplify the exponential amplification of the product;

[0010] (4) Due to the use of complementary primer pairs, the above methods will be limited by the size of the insertion or deletion fragment;

[0011] (5) Large primer PCR for site-directed mutagenesis, the annealing temperature (Tm) of the large primer and the outer primer must be significantly different, and the mutation efficiency cannot reach 100%

However, the method is cumbersome to operate, and multiple steps such as PCR, enzyme digestion, and ligation are required in sequence (Zhang ZQ, Xu K, Xin Y and Zhang ZY. An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes. Analytical Biochemistry. 2015, 476:26-28)

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