High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer

We developed a high-resolution melting (HRM) assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene using the intercalating dye SYTO 9. We tested 3 different cell lines with known KRAS mutations and then examined the sensitivity of mutation detection with the cell lines using 189 bp and 92 bp amplicons spanning codons 12 and 13. We then screened for KRAS mutations in 30 non-small cell lung cancer biopsies that had been previously sequenced for mutations in EGFR exons 18–21.Known KRAS mutations in cell lines (A549, HCT116 and RPMI8226) were readily detectable using HRM. The shorter 92 bp amplicon was more sensitive in detecting mutations than the 189 bp amplicon and was able to reliably detect as little as 5–6% of each cell line DNA diluted in normal DNA. Nine of the 30 non-small cell lung cancer biopsies had KRAS mutations detected by HRM analysis. The results were confirmed by standard sequencing. Mutations in KRAS and EGFR were mutually exclusive.HRM is a sensitive in-tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening of gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.With the advent of personalised medicine, there is a compelling need for rapid and accurate methods for detection of nucleic acid sequence changes in clinical specimens. An ideal technology should be sensitive enough to accommodate significant amounts of stromal and normal cell contamination, robust and simple enough to be readily implemented in a diagnostic laboratory, rapid enough to provide important therapeutic information in a clinical timeframe, and cost-efficient. High resolution melting (HRM) is an emerging technique for detection of nucleic acid sequence variation [1] that has enormous potential to meet these clinical demands. We have used detection of codon 12 and 13 mutations in the KRAS gene to establish that