High-Throughput Creation of a Whole-Genome Collection of Yeast Knockout Strains

  • Angela M. Chu
  • Ronald W. Davis
Part of the Methods in Molecular Biology™ book series (MIMB, volume 416)


Gene disruption methods have proved to be a valuable tool for studying gene function in yeast. Gene replacement with a drug-resistant cassette renders the disruption strain selectable and is stable against reversion. Polymerase chain reaction-generated deletion cassettes are designed with homology sequences that flank the target gene. These deletion cassettes also contain unique “molecular bar code” sequence tags. Methods to generate these mutant strains are scalable and facile, allowing for the production of a collection of systematic disruptions across the Saccharomyces cerevisiae genome. The deletion strains can be studied individually or pooled together and assayed in parallel utilizing the sequence tags with microarray-based methods.

Key Words

gene disruption homologous recombination sequence tags systematic disruption yeast deletion yeast knockout 


  1. 1.
    Goffeau, A., Barrell, B. G., Bussey, H., Davis, R. W., Dujon, B., Feldmann, H., et al. (1996) Life with 6000 genes. Science 274, 546–567.CrossRefPubMedGoogle Scholar
  2. 2.
    Suter, B., Auerbach, D., and Stagljar, I. (2006) Yeast-based functional genomics and pro-teomics technologies: the first 15 years and beyond. Biotechniques 40, 625–642.CrossRefPubMedGoogle Scholar
  3. 3.
    Guthrie, C., and Fink, G., eds. (1991) Guide to Yeast Genetics and Molecular Biology (Methods Enzymology, Vol 194). San Diego: Academic Press.Google Scholar
  4. 4.
    Shoemaker, D. D., Lashkari, D. A., Morris, D., Mittmann, M., and Davis, R. W. (1996) Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy. Nat. Genet. 14, 450–456.CrossRefPubMedGoogle Scholar
  5. 5.
    Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., et al. (1999) Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906.CrossRefPubMedGoogle Scholar
  6. 6.
    Gietz, R. D., and Woods, R. A. (1994) High efficiency transformation with lithium acetate. In: Johnston, J. R., ed. Molecular Genetics of Yeast, A Practical Approach. Oxford: IRL Press, pp. 121–134.Google Scholar
  7. 7.
    Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21, 3329–3330.CrossRefPubMedGoogle Scholar
  8. 8.
    Hong, E. L., Balakrishnan, R., Christie, K. R., Costanzo, M. C., Dwight, S. S., Engel, S. R., et al. Saccharomyces Genome Database. Available at Scholar
  9. 9.
    Wach, A., Brachat, A., Pohlmann, R., and Philippsen, P. (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808.CrossRefPubMedGoogle Scholar
  10. 10.
    Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132.CrossRefPubMedGoogle Scholar
  11. 11.
    Deutschbauer, A. M., Jaramillo, D. F., Proctor, M., Kumm, J., Hillenmeyer, M. E., Davis, R. W., et al. (2005) Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast. Genetics 169, 1915–1925.CrossRefPubMedGoogle Scholar
  12. 12.
    Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391.CrossRefPubMedGoogle Scholar
  13. 13.
    Rozen, S., and Skaletsky, H. J. (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz, S. and Misener, S, eds. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Totowa, NJ: Humana, pp. 365–386.Google Scholar
  14. 14.
    Zhang, Z., Schwartz, S., Wagner, L., and Miller, W. (2000) A greedy algorithm for aligning DNA sequences. J Comput. Biol. 7, 203–214.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Angela M. Chu
    • 1
  • Ronald W. Davis
    • 2
    • 3
  1. 1.Department of BiochemistryStanford University School of MedicineStanford
  2. 2.Departments of Biochemistry and GeneticsStanford University School of MedicineStanford
  3. 3.Stanford Genome Technology CenterPalo Alto

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