An efficient one-step site-directed and site-saturation mutagenesis protocol.
We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.
Zheng
Lei
L
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland.
Baumann
Ulrich
U
Reymond
Jean-Louis
JL
D017931:DNA Primers; D019020:Directed Molecular Evolution; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods
D017931:DNA Primers