Published online 19 July 2005
Methods Online |
A novel PCR strategy for high-efficiency, automated site-directed mutagenesis
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory Beijing 100875, People's Republic of China 1Department of Biochemistry, Queen's University Kingston, Canada ON K7L 3N6
*To whom correspondence should be addressed. Tel: +1 86 10 58807365; Fax: +1 86 10 58807365; Email: weiq{at}bnu.edu.cn
Received April 13, 2005. Revised June 13, 2005. Accepted July 1, 2005.
We have developed a novel three-primer, one-step PCR-based method for site-directed mutagenesis. This method takes advantage of the fact that template plasmid DNA cannot be efficiently denatured at its reannealing temperature (Tra), which is otherwise a troublesome problem in regular PCR. Two flanking primers and one mutagenic primer with different melting temperatures (Tm) are used together in a single PCR tube continuously without any intervention. A single-stranded mutagenic DNA (smDNA) is synthesized utilizing the high Tm mutagenic primer at a high annealing temperature, which prevents the priming of the low Tm primers (i.e. the two flanking primers). A megaprimer is then produced using this smDNA as the template at a denaturing temperature that prevents wild-type template DNA activity. The desired mutant DNA is then obtained by cycling again through these first two steps, resulting in a mutagenic efficiency of 100% in all tested cases. This highly automated method not only eliminates the necessity of any intermediate manipulation and accomplishes the mutagenesis process in a single round of PCR but, most notably, enables complete success of mutagenesis. This novel method is also both cost and time efficient and fully automated.