Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies -: art. no. e84

被引:14
作者
Merritt, J
DiTonno, JR
Mitra, RD
Church, GM
Edwards, JS [1 ]
机构
[1] Univ Delaware, Dept Chem Engn, Newark, DE 19716 USA
[2] Washington Univ, Dept Genet, St Louis, MO 63110 USA
[3] Harvard Univ, Sch Med, Dept Genet, Boston, MA 02115 USA
关键词
D O I
10.1093/nar/gng084
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We describe a strategy to analyze the impact of single nucleotide mutations on protein function. Our method utilizes a combination of yeast functional complementation, growth competition of mutant pools and polyacrylamide gel immobilized PCR. A system was constructed in which the yeast PGK1 gene was expressed from a plasmid-borne copy of the gene in a PGK1 deletion strain of Saccharomyces cerevisiae. Using this system, we demonstrated that the enrichment or depletion of PGK1 point mutants from a mixed culture was consistent with the expected results based on the isolated growth rates of the mutants. Enrichment or depletion of individual point mutants was shown to result from increases or decreases, respectively, in the specific activities of the encoded proteins. Further, we demonstrate the ability to analyze the functional effect of many individual point mutations in parallel. By functional complementation of yeast deletions with human homologs, our technique could be readily applied to the functional analysis of single nucleotide polymorphisms in human genes of medical interest.
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页数:8
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