Construction of mutant alleles in Saccharomyces cerevisiae without cloning: overview and the delitto perfetto method.

Traditionally, methods for introducing specific new mutations at target loci in the yeast genome have involved the preparation of disruption or gene-replacement cassettes via multiple cloning steps. Sequences used for targeting these cassettes or integrating vectors are typically several hundred base pairs long. A variety of newer methods rely on the design of custom PCR oligonucleotides containing shorter sequence tails (∼50 nt) for targeting the locus of interest. These techniques obviate the need for cloning steps and allow construction of mutagenesis cassettes by PCR amplification. Such cassettes may be used for gene deletion, epitope tagging, or site-specific mutagenesis. The strategies differ in several ways, most notably with respect to whether they allow reuse of the selection marker and whether extra sequences are left behind near the target locus. This unit presents a summary of methods for targeted mutagenesis of Saccharomyces cerevisiae loci without cloning, including PCR-based allele replacement, delitto perfetto, and MIRAGE. Next, a protocol is provided for the delitto perfetto PCR- and oligonucleotide-based mutagenesis method, which offers particular advantages for generating several different mutant alleles of the same gene.