New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis
作者： Yongzhen Xia , Wenqiao Chu , Qingsheng Qi , Luying Xun
DOI: 10.1093/NAR/GKU1189
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摘要: The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it incompatible with the proposed mechanism. complementary to linearly amplify a target plasmid products annealing produce double-stranded DNA molecules 5'-overhangs. overhang supposed form circular plasmids staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that PCR enzyme fills 5'-overhangs early cycles, and product then used as template for exponential amplification. linear homologous ends are joined generate desired mutations through recombination E. coli. correct understanding important improvements, guiding us use Phusion polymerase mutagenesis. did not plasmid, producing
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