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

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Microbial Gene Essentiality: Protocols and Bioinformatics

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 416))

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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.

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Author information

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, The High Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, MD

    Pamela B. Meluh, Daniel S. Yuan, Sharon Sookhai-Mahadeo, **aoling Wang & Jef D. Boeke

  2. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX

    Xuewen Pan

  3. McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD

    Carol Tiffany & Ou Chen

  4. United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD

    Brian D. Peyser

  5. Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD

    Rafael Irizarry

  6. Department of Molecular Biology and Genetics, McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD

    Forrest A. Spencer

Authors
  1. Pamela B. Meluh
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  2. Xuewen Pan
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  3. Daniel S. Yuan
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  4. Carol Tiffany
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  5. Ou Chen
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  6. Sharon Sookhai-Mahadeo
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  7. **aoling Wang
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  8. Brian D. Peyser
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  9. Rafael Irizarry
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  10. Forrest A. Spencer
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  11. Jef D. Boeke
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Editor information

Editors and Affiliations

  1. Burnham Institute for Medical Research, La Jolla, California

    Andrei L. Osterman

  2. Fellowship for Interpretation of Genomes (FIG), Burr Ridge, Illinois

    Svetlana Y. Gerdes

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© 2008 Humana Press Inc., a part of Springer Science+Business Media, LLC

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Meluh, P.B. et al. (2008). Analysis of Genetic Interactions on a Genome-Wide Scale in Budding Yeast: Diploid-Based Synthetic Lethality Analysis by Microarray. In: Osterman, A.L., Gerdes, S.Y. (eds) Microbial Gene Essentiality: Protocols and Bioinformatics. Methods in Molecular Biology™, vol 416. Humana Press. https://doi.org/10.1007/978-1-59745-321-9_15

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  • DOI: https://doi.org/10.1007/978-1-59745-321-9_15

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-378-7

  • Online ISBN: 978-1-59745-321-9

  • eBook Packages: Springer Protocols

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