Site-directed mutagenesis of single base in vitro using circular site-directed mutagenesis technology

Objective To mutate single base of hTSHR cDNA subcloned into pcDNA3.1 to normal base sequence by circular site-directed mutagenesis technology.Methods A pair of completely complementary primers with desired mutation was designed,circular plasmid was used as the template,the nicked mutational plasmids were obtained by PCR amplifying.Parental plasmids were digested by specific enzyme.Digested PCR products were transformed into E.coli competent cells for repairing nick and amplifying.Results As shown by DNA sequencing,desired mutation was successfully completed,and other bases of target gene were not mutated.Conclusion Circular site-directed mutagenesis technology is an efficient and rapid method of site-directed mutagenesis.Target base was successfully mutated by this method,which provides a basis for further study.