Asymmetric overlap extension PCR method for site-directed mutagenesis.

Overlap extension PCR (OE-PCR) has been widely used in site-directed mutagenesis. The original OE-PCR included two rounds of PCRs and required tedious steps to purify the first-round PCR product. By combining asymmetric PCR and overlap extension, a novel asymmetric overlap extension PCR (AOE-PCR) method has been developed. This method consists of two separate asymmetric PCRs of around 30 cycles and a single cycle of annealing and extension after directly mixing the first-round PCR products. AOE-PCR eliminates intermediate purification steps and amplification of wild-type template and requires fewer PCR cycles, and is, therefore, a much simpler and faster and more efficient site-directed mutagenesis method than the original OE-PCR approach.