Solid-Phase Hybridization Assay for Detection of Mutated Cancer DNA by Fluorescence

Abstract

We report a straightforward protocol for the detection of mutated DNA extracted from cancer cells. The assay combines a step-wise solid-phase hybridization and a readout by fluorescence emission. We detect a single-nucleotide polymorphism in two human oncogenes, BRAF and EGFR, and reach a limit of the detection of 300 pM by conventional fluorometry. The protocol described herein may be used as a foundation for development of automatic optimized assays capable for detection of mutant DNA and RNA in vitro and in cells.