A rapid and efficient one-step site-directed deletion, insertion, and substitution mutagenesis protocol

A rapid and efficient site-directed mutagenesis (SDM) protocol based on two separate polymerase chain reaction (PCR) amplifications and homologous recombination in Escherichia coli is described. This protocol can introduce deletions, substitutions, and insertions into any amplifiable site of the target genes by ligating two amplified DNA fragments into vectors. Compared with previously reported PCR-based SDM methods, our protocol significantly prevents primer dimerization even though partially complementary primers were used for PCR. The genome with the target gene was used directly as template, and DpnI was unnecessary. All of the procedures were performed within 24 h. The mutation frequencies of deletion, substitution, and insertion of the PEP4 (encode proteinase A) gene of Saccharomyces cerevisiae were nearly 100% using this new method. Thus, this method can potentially facilitate high-throughput genetic engineering and structure–function analyses and is useful for molecular biological research.