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Microbial Gene Essentiality: Protocols and Bioinformatics

Volume 416 of the series Methods in Molecular Biology™ pp 221-247

Analysis of Genetic Interactions on a Genome-Wide Scale in Budding Yeast: Diploid-Based Synthetic Lethality Analysis by Microarray

  • Pamela B. MeluhAffiliated withDepartment of Molecular Biology and Genetics, The High Throughput Biology Center, The Johns Hopkins University School of Medicine
  • , Xuewen PanAffiliated withDepartment of Biochemistry and Molecular Biology, Baylor College of Medicine
  • , Daniel S. YuanAffiliated withDepartment of Molecular Biology and Genetics, The High Throughput Biology Center, The Johns Hopkins University School of Medicine
  • , Carol TiffanyAffiliated withMcKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine
  • , Ou ChenAffiliated withMcKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine
  • , Sharon Sookhai-MahadeoAffiliated withDepartment of Molecular Biology and Genetics, The High Throughput Biology Center, The Johns Hopkins University School of Medicine
  • , Xiaoling WangAffiliated withDepartment of Molecular Biology and Genetics, The High Throughput Biology Center, The Johns Hopkins University School of Medicine
  • , Brian D. PeyserAffiliated withUnited States Army Medical Research Institute of Infectious Diseases, Fort Detrick
  • , Rafael IrizarryAffiliated withDepartment of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University
    • , Forrest A. SpencerAffiliated withDepartment of Molecular Biology and Genetics, McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine
    • , Jef D. BoekeAffiliated withDepartment of Molecular Biology and Genetics, The High Throughput Biology Center, The Johns Hopkins University School of Medicine

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Abstract

Comprehensive collections of open reading frame (ORF) deletion mutant strains exist for the budding yeast Saccharomyces cerevisiae. With great prescience, these strains were designed with short molecular bar codes or TAGs that uniquely mark each deletion allele, flanked by shared priming sequences. These features have enabled researchers to handle yeast mutant collections as complex pools of ∼6000 strains. The presence of any individual mutant within a pool can be assessed indirectly by measuring the relative abundance of its corresponding TAG(s) in genomic DNA prepared from the pool. This is readily accomplished by wholesale polymerase chain reaction (PCR) amplification of the TAGs using fluorescent oligonucleotide primers that recognize the common flanking sequences, followed by hybridization of the labeled PCR products to a TAG oligonucleotide microarray. Here we describe a method—diploid-based synthetic lethality analysis by microarray (dSLAM)—whereby such pools can be manipulated to rapidly construct and assess the fitness of 6000 double-mutant strains in a single experiment. Analysis of double-mutant strains is of growing importance in defining the spectrum of essential cellular functionalities and in understanding how these functionalities interrelate.

Key Words

genetic interaction molecular barcode oligonucleotide microarray SLAM synthetic lethality yeast knock-out strains