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Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high‐resolution melting analysis
Author(s) -
Kristensen Lasse S.,
Andersen Gitte B.,
Hager Henrik,
Hansen Lise Lotte
Publication year - 2012
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.21598
Subject(s) - high resolution melt , amplicon , biology , primer (cosmetics) , mutation , melting curve analysis , melting temperature , cold pcr , genetics , microbiology and biotechnology , mutation testing , mutant , polymerase chain reaction , point mutation , genotyping , computational biology , genotype , gene , materials science , chemistry , organic chemistry , composite material
Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild‐type and mutated sequences simultaneously using a three‐primer system. A mutation‐specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild‐type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high‐resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD‐PCR). For proof‐of‐concept, we have designed CADMA assays for clinically relevant BRAF , EGFR , KRAS , and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild‐type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis. Hum Mutat 33:264–271, 2012. © 2011 Wiley Periodicals, Inc.