New insights into the QuikChange™ process guide the use of Phusion DNA polymerase for site-directed mutagenesis.
The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5'-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for site-directed mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for site-directed mutagenesis.
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, P. R. China.
Yongzhen
Y
Xia
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, P. R. China.
Wenqiao
W
Chu
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, P. R. China.
Qingsheng
Q
Qi
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, P. R. China School of Molecular Biosciences, Washington State University, Pullman, WA 99164-7520, USA luying_xun@vetmed.wsu.edu.
Luying
L
Xun
D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
D017931:DNA Primers; D004247:DNA; D004259:DNA-Directed DNA Polymerase