MutS based SNP detection, genome scanning and rare sequence enrichment The goal of this project is to develop MutS based assays for SNP detection, rare sequence detection, rare sequence isolation and genome scanning for SNP discovery. The approach will be to use end-blocking of heteroduplexes, which has been shown to improve the stability of MutS binding. Initial experiments will involve SNP detection and rare SNP detection and isolation using synthetic oligonucleotides and will utilize Gene Check's Immobilized Mismatch Binding Protein method of SNP detection. These experiments will also determine if end- blocking improves MutS binding to mismatches that are poorly bound in vivo and in vitro without end-blocking. Three different end-blocks will be tested (four-way DNA junctions, streptavidin/biotin complexes and antibody/biotin complexes)and MutS will be obtained from three different bacteria (E. coli, T. aquaticus and T. thermophilis). Successful conditions will be applied to PCR amplicons for SNP and rare SNP detection by amplifying genomic DNA of known genotypes mixed in various ratios. Finally, bacterial genomic DNA will be used in experiments testing end-blocking in applications for genomic scanning for unknown SNPs and for direct genotyping of known SNPs without amplification. MutS based SNP detection, genome scanning and rare sequence enrichment Development of a MutS based method of SNP detection and discovery and rare SNP and sequence detection and isolation will help meet an increasing need for SNP discovery and whole genome scanning in the move toward personalized medicine. The ability to detect rare SNPs and sequences will have immediate clinical impact, both for humans in such areas as diagnostic scanning for rare mutations among normal cells as in tumor margins, and in veterinary diagnostics by allowing rapid and inexpensive analysis of pooled samples for infectious agents and genetic diseases or production traits. ? ? ?
