title: Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products.
abstract: 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.
authors.affiliation: Institute of Human Genetics, University of Aarhus, The Bartholin Building, Aarhus C, Denmark. lasse@humgen.au.dk
authors.forename: Lasse S
authors.initials: LS
authors.lastname: Kristensen
authors.forename: Iben L
authors.initials: IL
authors.lastname: Daugaard
authors.forename: Mariann
authors.initials: M
authors.lastname: Christensen
authors.forename: Stephen
authors.initials: S
authors.lastname: Hamilton-Dutoit
authors.forename: Henrik
authors.initials: H
authors.lastname: Hager
authors.forename: Lise Lotte
authors.initials: LL
authors.lastname: Hansen
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D003080:Cold Temperature; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000378:metabolism; D005091:Exons / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D011544:Pseudogenes / Q000235:genetics; D015203:Reproducibility of Results; D018631:ras Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins