Simultaneous Genotyping of Multiple Somatic Mutations by Using a Clamping PNA and PNA Detection Probes

It has been very difficult to detect and quantify multiple somatic mutations simultaneously in single-tube qPCR. Here, a novel method for simultaneous detection of multiple mutations and a melting curve analysis was developed by using clamping PNA and detection PNA probes. Each PNA probe was designed to have a specific melting temperature by the introduction of gamma-PNA monomer. This technique was successfully applied to the detection of six genotypes in two different mutations of K-RAS at the same time. Such simultaneous analysis of an amplified curve and a melting curve in qPCR can be widely used for the early diagnosis of cancer and determining the prognosis of drug treatments.