High-Throughput Creation of a Whole-Genome Collection of Yeast Knockout Strains
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 Wordsgene disruption homologous recombination sequence tags systematic disruption yeast deletion yeast knockout
- 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
- 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
- 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 ftp://ftp.yeastgenome.org/yeast/.Google Scholar
- 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
- 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