Abstract
The QuikChangeTM 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 QuikChangeTM 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.
Highlights
Polymerase chain reaction (PCR)-based site-directed mutagenesis is an essential technique in molecular, biochemical and genetic studies
The QuikChangeTM method uses PfuTurbo DNA polymerase to amplify a circular plasmid with a pair of complementary primers, which are completely overlapping to each other (Supplementary Figure S1)
The PCR progresses via linear amplification for the production of singlestranded DNA molecules that anneal to generate the circular plasmid with staggered single-stranded DNA breaks
Summary
Polymerase chain reaction (PCR)-based site-directed mutagenesis is an essential technique in molecular, biochemical and genetic studies. Several methods have been developed, including overlap extension PCR, megaprimer PCR, QuikChangeTM site-directed mutagenesis [1,2,3,4,5], among which the QuikChangeTM method (Agilent Technologies, La Jolla, CA, USA) is widely used [6]. The PCR progresses via linear amplification for the production of singlestranded DNA molecules that anneal to generate the circular plasmid with staggered single-stranded DNA breaks (the QuikChangeTM manual; Agilent Technologies). The proposed process is similar to a process done in two separate PCR reactions before mixing the products to generate the circle DNA with staggered single-stranded DNA breaks [7]. After DpnI digestion of the methylated plasmid template and transformation into Escherichia coli, the host cells repair the breaks to yield the plasmid with the desired mutation
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