Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies.
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.
Merritt
Joshua
J
Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.
DiTonno
Jason R
JR
Mitra
Robi D
RD
Church
George M
GM
Edwards
Jeremy S
JS
D001704:Biopolymers / Q000032:analysis; D003198:Computer Simulation; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008954:Models, Biological; D010735:Phosphoglycerate Kinase / Q000235:genetics / Q000502:physiology; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology; D014170:Transformation, Genetic
D001704:Biopolymers; D004247:DNA; D010735:Phosphoglycerate Kinase