The publication of the yeast genome sequence opened new opportunities for yeast geneticists. Knowing the DNA sequence of the yeast genome, geneticists could now take advantage of the high frequency with which yeast exchange genes by homologous recombination to generate mutants of their own design. Homologous recombination normally occurs during meiosis and during certain kinds of DNA repair. During homologous recombination, two closely related DNA sequences align with one another, breaks appear in the DNA molecules and strand exchange occurs when the breaks are repaired. Homologous recombination allows researchers
to replace a chromosomal gene with a DNA construct of their own design. To use this strategy, investigators first construct a replacement cassette in which a marker gene is flanked by sequences that are identical to chromosomal DNA sequences at either side of the the yeast target gene. The sequence is then introduced into cells by chemical transformation (Chapter 12) or by electroporation. Transformed cells that have incorporated the replacement cassettes into chromosomal DNA can be identified by selecting for the marker gene in the replacement cassette.
The Saccharomyces Genome Deletion Project (SGDP) used this approach to systematically replace each of the predicted ORFs in the S. cerevisiae genome with a kanamycin resistance (KANR) gene. For each ORF, researchers used a series of PCR reactions (below) to construct cassettes in which the KANR gene was flanked by short DNA sequences that occur upstream and downstream of the targeted ORF on the S. cerevisiae chromosome. The cassettes were then used to transform the BY4742 strain (Brachmann et al., 1998). Strains that had incorporated the KANR gene were selected on plates containing analogs of kanamycin (Winzeler et al., 1999).
The S. cerevisiae strains that we are using in our experiments are part of the SGDP collection of deletion mutants. In this lab, you will use PCR to identify which MET genes have been disrupted in your strains. The PCR primers were designed and tested by the SGDP to verify strain identities. Available primers include two gene-specific primers (GSP) for each of the METgenes. One of the GSPs, GSP Primer A, is located 200-400 bp upstream of the initiation codon. The second GSP, GSP Primer B, is an antisense primer that binds within the ORF. We also have an antisense primer that binds 250 bp within the KANR gene (KAN Primer B).