Long Distance Multiple-Site Directed Plasmid Mutagenesis by One-Step PCR Using Non-overlapped Primers

Site-directed mutagenesis is a very important technique in molecular biological researches. We have developed a new method for long distance multiple-site plasmid mutation by one-step PCR using non-overlap primers. These primers were carefully designed and contained desired mutations in the middle of the primers flanked with 18–25 bp of correct sequence. One pair of the primers was able to generate a short megaprimer. Decreases in the concentrations of these primers increased efficiency of the multiple-site plasmid mutation. All of the mutant PCRs were performed at a common annealing temperature at 55 °C. This method could be widely used in all multiple-site plasmid mutations.