Site-directed Mutagenesis Based on Overlap Extension PCR

[Objective] To establish an efficient,convenient and economical method for site-directed mutagenesis.[Method] The target mutation was introduced into primers designed by DNAMAN5.0 software.Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator(Reteplase).The mutation gene cloned it into pEASY-blunt simple cloning vector for sequencing.[Result] The sequencing results showed that three site mutations were fully consistent with the expected results(10th site had been added a base-pair of A,C had been changed into G at 137th site,G had been changed into A at 686th site).Three site mutations were introduced by using overlap extension PCR on one-step.The overall rate of obtaining the mutant sites was 100%.Site-directed mutagenesis will clone the recombinant Human Tissue type plasminogen activator and laid the basis for the functional study.[Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient,convenient and economical DNA-directed mutagenesis method.