Signature-based analysis of MET proto-oncogene mutations using DHPLC

Research tools which improve mutation detection, SNP discovery, and allele characterization will facilitate studies of cancer, inherited disease, and genomic evolution. Denaturing High-Performance Liquid Chromatography (DHPLC ) is a recently developed methodology for detection of heteroduplexes forme d in DNA samples containing mismatches between wild type and mutant strands . In an effort to develop a rapid, sensitive mutation detection method for studies of families with inherited kidney cancer, we evaluated DHPLC for de tection and analysis of MET protooncogene mutations in papillary renal carc inomas (PRC), We found DHPLC to be 100% accurate in detecting 15 known dise ase-associated MET mutations. Significantly, each MET mutation and two nove l SNPs generated a characteristic chromatographic profile or signature with reproducible distinguishing features. Standardization of DHPLC reagents an d improved methods design were critical to the reliability and accuracy of mutation prediction. Improvements included addition of a 75% acetonitrile w ash followed by a rejuvenating gradient, and detailed analysis of signature shape, retention time (RT), RT differences (Delta RT), and temperature-dep endent (melt) profiling, We used signatures to predict mutations in new PRC samples, mutation carriers in asymptomatic hereditary PRC family members, and in a blind study of previously characterized DNAs. Application to SNP d iscovery is discussed, Hum Mutat 16:68-76, 2000, Published 2000 (C) Wiley-L iss, Inc.(dagger)