Nature Biotechnology
17, 804 - 807 (1999)
doi:10.1038/11751
Detection of PCR products using self-probing amplicons and fluorescence
David Whitcombe1, Jane Theaker1, Simon P. Guy1, Tom Brown2
& Steve Little11
AstraZeneca Diagnostics, Gadbrook Park
, Rudheath, Northwich, Cheshire CW9 7RA
, UK. 2
Department of Chemistry, University of Southampton
, Highfield, Southampton SO17 1BJ,
UK.
Correspondence should be addressed to David Whitcombe david.whitcombe@diagnostics.zeneca.comHomogeneous assaysfluorescent probesdiagnosticsPCRARMSsingle-tube genotypingmultiplexingMolecular 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.
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