A one-step polymerase chain reaction site-directed mutagenesis method for large gene-cassettes with high efficiency, yield, and fidelity.

A one-step three-stage efficient PCR method for site-directed mutagenesis has been developed. This method involves one PCR reaction with no need to add more reagents during this reaction or to do a second PCR reaction. This method utilizes a three-stage PCR cycling profile, a ddNTP-blocked restriction endonuclease fragment, and Pfu DNA polymerase. This method allows the amplification of at least 2 kb of final mutant product via an intermediate megaprimer as large as 1.3 kb with high fidelity, high efficiency, high yield, and high success rate. A 3.2-kb large final mutant product has also been obtained by using Taq and Taq Extender (Stratagene) instead of Pfu. This method is particularly useful when there are no unique restriction sites in the gene of interest to reduce the large sizes of the final mutant product and/or the megaprimer.