Abstract
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.
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References
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.
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.
Guthrie, C., and Fink, G., eds. (1991) Guide to Yeast Genetics and Molecular Biology (Methods Enzymology, Vol 194). San Diego: Academic Press.
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.
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.
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.
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.
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/.
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.
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.
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.
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.
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.
Zhang, Z., Schwartz, S., Wagner, L., and Miller, W. (2000) A greedy algorithm for aligning DNA sequences. J Comput. Biol. 7, 203–214.
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Chu, A.M., Davis, R.W. (2008). High-Throughput Creation of a Whole-Genome Collection of Yeast Knockout Strains. 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_14
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DOI: https://doi.org/10.1007/978-1-59745-321-9_14
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