Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries.
We have developed a new method, systematic hybrid loss of heterozygosity, to facilitate genomic screens utilizing the yeast gene deletion library. Screening is performed using hybrid diploid strains produced through mating the library haploids with strains from a different genetic background, to minimize the contribution of unpredicted recessive genetic factors present in the individual library strains. We utilize a set of strains where each contains a conditional centromere construct on one of the 16 yeast chromosomes that allows the destabilization and selectable loss of that chromosome. After mating a library gene deletion haploid to such a conditional centromere strain, which corresponds to the chromosome carrying the gene deletion, loss of heterozygosity (LOH) at the gene deletion locus can be generated in these otherwise hybrid diploids. The use of hybrid diploid strains permits complementation of any spurious recessive mutations in the library strain, facilitating attribution of the observed phenotype to the documented gene deletion and dramatically reducing false positive results commonly obtained in library screens. The systematic hybrid LOH method can be applied to virtually any screen utilizing the yeast non-essential gene deletion library and is particularly useful for screens requiring the introduction of a genetic assay into the library strains.
Alvaro
David
D
Department of Genetics and Development, Columbia University Medical Center, New York, NY 10032, USA.
Sunjevaric
Ivana
I
Reid
Robert J D
RJ
Lisby
Michael
M
Stillman
David J
DJ
Rothstein
Rodney
R
D001426:Bacterial Proteins / Q000235:genetics; D002869:Chromosome Aberrations; D004171:Diploidy; D017353:Gene Deletion; D005820:Genetic Testing / Q000379:methods*; D016681:Genome, Fungal; D015698:Genomic Library; D006238:Haploidy; D019656:Loss of Heterozygosity; D008164:Luminescent Proteins / Q000235:genetics; D009154:Mutation; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; C054562:yellow fluorescent protein, Bacteria