Unlabeled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in pancreatic adenocarcinoma tissues.

BACKGROUND The aim of our study was to establish an unlabeled-probe high-resolution melting (HRM) approach to the detection of Kirsten RAS (KRAS) codon 12 and 13 mutations in pancreatic adenocarcinoma (PA) tissues as a novel and effective diagnostic technique. METHODS We tested the sensitivity and specificity of this genotyping approach in cell lines with known KRAS mutations using 166 bp amplicons and 37 bp wild-type probe to detect KRAS codon 12 and 13 mutations. We screened 49 PA tissues to be subsequently sequenced to confirm the mutations. Simultaneously, we tested the specimens using Sanger sequencing and then used target-DNA cloning and sequencing for verification. RESULTS It was found that unlabeled-probe HRM was reliable in detecting 3% of mutant cell lines DNA diluted with that of the wild-type, whereas Sanger sequencing could only discriminate 20% mutant cell ratios. In detecting 49 specimens, the former was capable of detecting 23 mutations (46.9%); and the latter could observe 15 (30.6%). For further verification, T-A DNA cloning and sequencing was applied to the differences, with the results matching those of the unlabeled-probe HRM. CONCLUSIONS It was concluded that the unlabeled-probe HRM approach can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in diagnosing and treating PA.