Detection of PCR products using self-probing amplicons and fluorescence

作者：Whitcombe D^*; Theaker J; Guy SP; Brown T; Little S

来源：Nature Biotechnology, 1999, 17(8): 804-807.

DOI：10.1038/11751

• homogeneous assays
• fluorescent probes
• diagnostics
• PCR
• ARMS
• single-tube genotyping
• multiplexing

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.

• 出版日期1999-8