91 results about "Targeted Mutation" patented technology

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%.