Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high‐resolution melting analysis

摘要: Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, individualized patient treatment. However, the majority molecular methodologies that have been developed with aim increasing sensitivity testing drawbacks terms specificity, convenience, or costs. Here, we established a new method, Competitive Amplification Differentially Melting Amplicons (CADMA), which allows very sensitive all types. The principle method to amplify wild-type mutated sequences simultaneously using three-primer system. A mutation-specific primer designed introduce melting temperature decreasing mutations resulting amplicon, while second overlapping both sequences. When combined third common becomes possible, when high-resolution (HRM) as platform. introduction amplicon also for further enrichment by fast coamplification at lower denaturation PCR (COLD-PCR). For proof-of-concept, CADMA assays clinically relevant BRAF, EGFR, KRAS, PIK3CA mutations, are to, between 0.025% 0.25%, alleles background. In conclusion, enables highly HRM analysis.