Manipulating the yeast genome: deletion, mutation, and tagging by PCR

Saccharomyces cerevisiae is an ideal model eukaryotic system for the systematic analysis of gene function due to the ease and precision with which its genome can be manipulated. The ability of budding yeast to undergo efficient homologous recombination with short stretches of sequence homology has led to an explosion of PCR-based methods to delete and mutate yeast genes and to create fusions to epitope tags and fluorescent proteins. Here, we describe commonly used methods to generate gene deletions, to integrate mutated versions of a gene into the yeast genome, and to construct N- and C-terminal gene fusions. Using a high-efficiency yeast transformation protocol, DNA fragments with as little as 40 bp of homology can accurately target integration into a particular region of the yeast genome.