Objective: The aim of this study was to establish a differential high-resolution melting analysis for the detection of K-ras codons 12 and 13 mutations in pancreatic cancer tissue.
Methods: We tested the sensitivity of this genotyping approach in cell lines with known K-ras mutations using 163- and 59-base pair (bp) amplicons spanning codons 12 and 13. We then screened 50 pancreatic cancer tissues, which were subsequently sequenced to confirm mutations discovered in K-ras codons 12 and 13.
Results: High-resolution melting analysis was more sensitive in detecting mutations than standard sequencing and was able to reliably detect as little as 10% of mutant cell line DNA diluted in wild-type cell line DNA. Compared with the 163-bp amplicons, the shorter 59-bp amplicons displayed a higher sensitivity, even at 3% to 5% dilution.
Conclusions: High-resolution melting analysis is a sensitive and accurate screening methodology for K-ras codons 12 and 13 mutations in clinical samples. The accuracy and sensitivity of high-resolution melting analysis can provide appropriate and cost-effective therapeutic choices for physicians.