The Saccharomyces Genome Project has revealed the presence of more than 6000 open reading frames (ORFs) in the S. cerevisiae genome. Approximately one third of these ORFs currently have no known function four years after their discovery. The goal of the Saccharomyces Genome Deletion Project is to generate as complete a set as possible of yeast deletion strains with the overall goal of assigning function to the ORFs through phenotypic analysis of the mutants.
The method used was a PCR-based gene deletion strategy to generate a start- to stop- codon deletion of each of the ORFs in the yeast genome. As part of the deletion process, each gene disruption was replaced with a KanMX module and uniquely tagged with one or two 20mer sequence(s) . The presence of the tags can be detected via hybridization to a high-density oligonucleotide array, enabling growth phenotypes of individual strains to be analyzed in parallel .
Nearly all ORFs larger than 100 codons were disrupted; highly similar ORFs were not attempted (~3%). Four different mutant collections have been generated: haploids of both mating types, homozygous diploids for non-essential genes, and heterozygous diploids, which contain the essential and non essential ORFs. Our inital results show that 18.7% of the genes are essential for growth on rich gluose media and approximately 15% of the homozygous diploid disruptions cause slow growth in this type of media.
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