Simultaneous mutations up to six distal sites using a phosphorylation-free and ligase-free polymerase chain reaction-based mutagenesis.
In this study, we present an efficient phosphorylation-free and ligase-free PCR-based multiple site-directed mutagenesis that allows simultaneous mutations up to six distal sites. This method could be extended to any plasmid DNA that is isolated from dam(+)Escherichia coli strains, and the results showed that the simultaneously mutagenic efficiencies of quadruple mutation and sextuple mutation were up to 80% and 40%, respectively.
Tseng
Wen-Chi
WC
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan.
Lin
Jing-Wei
JW
Hung
Xing-Gung
XG
Fang
Tsuei-Yun
TY
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000191:economics / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*