Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis.
By combining asymmetric PCR and overlap extension, we developed a novel asymmetric overlap extension PCR (AOE-PCR) method for site-directed mutagenesis which bypassed the need for intermediate purification and excluded the amplification of a wild-type template. This method was used to introduce single base mutations into a small GTPase gene from cotton and to simultaneously introduce two mutations just by repeating this method using the first round AOE-PCR products as template. Our results suggested that the AOE-PCR method represents a valuable improvement of the original overlap extension PCR for site-directed mutagenesis.
Xiao
Yue-Hua
YH
Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Chongqing, PR China.
Yin
Meng-Hui
MH
Hou
Lei
L
Luo
Ming
M
Pei
Yan
Y
D003368:Gossypium / Q000235:genetics; D008957:Models, Genetic; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010940:Plant Proteins / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D010940:Plant Proteins; D020559:Monomeric GTP-Binding Proteins