Detection of PCR products using self-probing amplicons and fluorescence

Molecular diagnostics is progressing from low-throughput, heterogeneous, mo stly manual technologies to higher throughput, closed-tube, and automated m ethods. Fluorescence is the favored signaling technology for such assays, a nd a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule, In these methods, dual-labeled probes hybrid ize to an amplicon and changes in the quenching of the fluorophore are dete cted. We describe a new technology that is simple to use, gives highly spec ific information, and avoids the major difficulties of the alternative meth ods, It uses a primer with an integral tail that is used to probe an extens ion product of the primer, The probing of a target sequence is thereby conv erted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.