PCR AND RECOMBINATION FOR GENOMIC ANALYSIS IN YEAST

Columbia University Health Sciences, New York NY

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Abstract

The long-term research objective is to bring genomic analysis to many researchers using a set of common molecular biology techniques. A complete genome sequence exists for several bacterial species and two eukaryotes. Genomes of other model organisms and the human genome will be sequenced soon after the turn of the century. The challenge for researchers is to find creative ways to make use of this vast amount of information. During the fellowship, I will develop and test PCR and recombination techniques necessary to rapidly assemble gene replacement vectors directly in the yeast Saccharomyces cerevisiae. The gene replacement vectors will be portable by a mating reaction, meaning they can be rapidly moved from a donor set of yeast strains to a laboratory strain of interest. Gene disruption, or "knockout," vectors and allele replacement vectors will be developed so that any yeast gene can be disrupted or replaced by a conditional allele. Each type of vector will use a selectable marker that is recyclable, permitting multiple rounds of gene replacement into a single strain. Knockout vectors will also introduce a unique DNA sequence tag to identify each knockout strain. The culmination of these techniques will be a genome-wide library that can be rapidly disseminated through the research community to aid genome research.