An Improved Site-Directed Mutagenesis Procedure and Application for Identification of Mutant Clones

作者：Chunyu Li, Qingming Sun, Shi Chen, Guiming Deng, Cunwu Zuo, Qian Ye, Jing Yang, Ganjun Yi · 发表于：Biotechnology & Biotechnological Equipment · 年份：2012 · DOI：10.5504/bbeq.2011.0140 · 研究领域：Plant Genetic and Mutation Studies、Plant tissue culture and regeneration、Chromosomal and Genetic Variations

A number of molecular tools have been developed to study pathogenicity in fungal phytopathogens, including Fusarium species. Here, we reported an improved method for site-directed mutagenesis to create constructs for using in the analysis of fungal gene function. The procedure combining overlap-extension PCR (OE-PCR) with S1 nuclease mismatch cleavage, which we refer to as OE-PCR-S1, is more time- and labor-efficient. We tested our method by deleting three bases from the pathogenic gene Fowl (EU795421) and the resulting mutant DNA was inserted into an expression vector before transformation into Foc for functional domain analysis. The overall rate of mutant site production was 100% and the entire process was completed in less than two days. These results demonstrate that this method is a powerful tool for studying the functions of pathogenic genes and genetic engineering.