Site-directed mutagenesis of Saccharomyces cerevisiae genome using mismatch PCR product

Site-directed mutagenesis (SDM) is an important tool in molecular biology, especially in genetics and protein engineering. It can alter specific amino acid residues by changing the codons of a gene, thus allowing to explore the role of specific amino acid residues in the structure and function of the aim protein. Here, we developed a method for constructing SDM of the Saccharomyces cerevisiae genome based on homologous recombination in vivo using mismatch PCR product. In this method, mutations were designed in the PCR primers. After transforming the mismatch PCR product into yeast, positive colonies were screened via colony PCR with a verification primer whose 3' end were the mutated bases. To avoid false positive results in the screening, the annealing temperature of the colony PCR was optimized via temperature gradient PCR. This method was applied to the Exo70 subunit of the tethering complex exocyst, which is an effector of Rho3 and critical for localization of exocyst to the cell membrane. Finally, we successfully introduced the desired mutations in Exo70 within three days, showing that this method is simple, fast and feasible for SDM of yeast genome.