Signature-based analysis of MET proto-oncogene mutations using DHPLC
✍ Scribed by Michael L. Nickerson; Gregor Weirich; Berton Zbar; Laura S. Schmidt
- Book ID
- 101344036
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 335 KB
- Volume
- 16
- Category
- Article
- ISSN
- 1059-7794
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✦ Synopsis
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 formed 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 detection and analysis of MET protooncogene mutations in papillary renal carcinomas (PRC). We found DHPLC to be 100% accurate in detecting 15 known disease-associated MET mutations. Significantly, each MET mutation and two novel SNPs generated a characteristic chromatographic profile or signature with reproducible distinguishing features. Standardization of DHPLC reagents and improved methods design were critical to the reliability and accuracy of mutation prediction. Improvements included addition of a 75% acetonitrile wash followed by a rejuvenating gradient, and detailed analysis of signature shape, retention time (RT), RT differences (DRT), and temperature-dependent (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 discovery is discussed.