New insights into the QuikChange™ process guide the use of Phusion DNA polymerase for site-directed mutagenesis.

ABSTRACT: 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 wi

SUBMITTER: Xia Y 

PROVIDER: S-EPMC4333370 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature