[Tm analysis method using a quenching probe is a simple and rapid way to simultaneously detect KRAS and BRAF...

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that, on ligand binding, triggers the RAS-RAF-MAPK signaling pathway which is mainly associated with cell proliferation. Drugs targeting EGFR have recently entered clinical practice and have proven to be effective in providing clinical benefits. However, the presence of mutated KRAS alleles in cancer is a predictive marker of anti-EGFR drug resistance. Similarly, alterations of other members of the RAS-RAF-MAPK signaling pathway may also be such predictive markers, especially the BRAF mutation in colorectal cancer. Therefore, the identification of KRAS and BRAF mutations may be important in predicting resistance to EGFR-targeted therapies. We looked at the Tm analysis for simultaneous detection of KRAS and BRAF mutations using a quenching probe. The oligonucleotide probes modified with certain fluorescent dyes at 5'-end cytosine are quenched by their interaction with a uniquely positioned guanine. When the probe is hybridized with target DNA, its fluorescence is quenched by the guanine in the target DNA. However, as the temperature is raised, perfect match probes and miss match probes dissociate at different temperatures. Dissociated probes generate fluorescence. This simple and rapid method for simultaneous detection of these mutations is useful in clinical practice.