Detection of DNA mutations based on analysis of multiple wavelength excitation/emission fluorescence kinetics curves in real-time PCR

The key method for therapies of various cancer types could be the molecular-targeted therapy, based on individual gene profile for each patient. One of the main procedures used for genetic testing is the real-time polymerase chain reaction (real-time PCR).Physical principle behind real-time PCR procedure is the fluorescence. Fluorescence labeled probes (primers) is attached to quenchers. Upon reaction of polymerization, quenchers are removed, and the fluorescence emission intensity increases in time.Emission spectra shape and its maximum position can differ if the fluorophore was present in different microenvironment. That property is widely exploited in fluorescence spectroscopy and chromatography.This paper, for the first time, describes utilization of full spectroscopic potential of multichannel excitation/emission filter sets in real-time PCR device. Instead of monitoring fluorescence intensity in time for a single fluorescence emission channel, the ratio values of three different kinetics curves were calculated and analyzed by applying k-means clustering and dendrogram analysis.Obtained results have shown that described analytical improvement provides identification of nine different groups of mutations if the commercial QIAGEN® EGFR PCR Kit was used. Method can be applied to any kit, capable to simultaneously detect several different mutations.