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A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method

2007; Elsevier BV; Volume: 375; Issue: 2 Linguagem: Inglês

10.1016/j.ab.2007.12.013

1096-0309

Wen‐Chi Tseng, Jing-Wei Lin, Tsen-Yun Wei, Tsuei‐Yun Fang,

Genomics and Phylogenetic Studies

In this study, we report a novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method modified from the QuikChange site-directed mutagenesis (QCM). One mutagenic oligonucleotide and one universal flanking primer were used to produce the complementary megaprimers that were then used to amplify the whole plasmid template. This method yields a mutagenesis efficiency (∼90%) similar to that of QCM but uses only one mutagenic oligonucleotide instead of two of them, and the length of the oligonucleotide could be shorter. This method can be further extended to double mutations that are located at distant sites by using two mutagenic oligonucleotides and even to site saturation mutagenesis by introducing randomized codons.