A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method.
In this study, we report a novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method modified from the QuikChange site-directed mutagenesis (QCM). One mutagenic oligonucleotide and one universal flanking primer were used to produce the complementary megaprimers that were then used to amplify the whole plasmid template. This method yields a mutagenesis efficiency ( approximately 90%) similar to that of QCM but uses only one mutagenic oligonucleotide instead of two of them, and the length of the oligonucleotide could be shorter. This method can be further extended to double mutations that are located at distant sites by using two mutagenic oligonucleotides and even to site saturation mutagenesis by introducing randomized codons.
Tseng
Wen-Chi
WC
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan.
Lin
Jing-Wei
JW
Wei
Tsen-Yun
TY
Fang
Tsuei-Yun
TY
D017931:DNA Primers / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D008025:Ligases; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D009841:Oligonucleotides; D008025:Ligases