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

Original publication

DOI

10.1038/11751

Type

Journal article

Journal

Nat Biotechnol

Publication Date

08/1999

Volume

17

Pages

804 - 807

Keywords

BRCA2 Protein, Base Sequence, DNA Primers, Fluorescence, Kinetics, Molecular Probes, Neoplasm Proteins, Polymerase Chain Reaction, Thermodynamics, Transcription Factors