Site-directed mutagenesis based on overlap extension PCR.

Objective:To establish a fast, saving method for site-directed mutagenesis. Methods:Overlap extension PCR was used. Briefly, target mutation was introduced into primers, and the two previous PCR products were used as template for the third PCR. The final PCR segment with target mutant was then cloned into pMD? 18-T vector for sequencing. Results:DNA sequencing showed that the target site ATTGG had been changed into ATTTT. Conclusion:Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is a fast and saving method.