Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products.

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low-level tumor specific KRAS mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation-containing templates based on the lower melting temperature of mutant homoduplexes versus wild-type homoduplexes. We have developed a fast COLD-PCR and high-resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD-PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen(®) KRAS mutation kit. The sensitivity was increased by 5- to 100-fold for melting temperature decreasing mutations when using COLD-PCR compared to standard PCR. Mutations, undetectable by the TheraScreen(®) kit in clinical samples, were detected by COLD-PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD-PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments.