A novel PCR strategy for high-efficiency, automated site-directed mutagenesis.
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 (T(ra)), which is otherwise a troublesome problem in regular PCR. Two flanking primers and one mutagenic primer with different melting temperatures (T(m)) are used together in a single PCR tube continuously without any intervention. A single-stranded mutagenic DNA (smDNA) is synthesized utilizing the high T(m) mutagenic primer at a high annealing temperature, which prevents the priming of the low T(m) 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.
Wu
Wu
W
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory Beijing 100875, People's Republic of China.
Jia
Zongchao
Z
Liu
Ping
P
Xie
Zhigang
Z
Wei
Qun
Q
D017931:DNA Primers; D016297:Mutagenesis, Site-Directed; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
D017931:DNA Primers