A separation-free assay for the detection of mutations: combination of homogeneous time-resolved fluorescence and minisequencing

Single nucleotide primer extension reaction has been widely used in DNA testing, and several detection methods based on this core allelic discrimination have been developed. Most of the reported formats are based on a two step protocol involving first, a liquid phase extension reaction, then a physical separation process (chromatography, electrophoresis, capture on solid support, mass spectrometry). Here we describe a new strategy based on homogeneous time-resolved fluorescence (HTRF), which does not involve any separation process and which allows a simple "mix and measure" protocol. In this approach, a 5'-(europium) cryptate-labeled primer is elongated by a biotinylated dideoxynucleoside-triphosphate, followed by the addition of a streptavidin-acceptor conjugate, which gives rise to a long-life fluorescence resonance energy transfer (FRET) signal between the cryptate donor and the acceptor. We present the development of HTRF technology as applied to the diagnosis of tumor suppressor gene p53 (TP53) mutations, and its application to the analysis of genomic DNA from human tumoral samples. The sensitivity of the reported method is compared to the corresponding fluorescent polarization assay.