Detection of PCR products using self-probing amplicons and fluorescence.
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.
Whitcombe
D
D
AstraZeneca Diagnostics, Gadbrook Park, Rudheath, Northwich, Cheshire CW9 7RA, UK. david.whitcombe@diagnostics.zeneca.com
Theaker
J
J
Guy
S P
SP
Brown
T
T
Little
S
S
D024682:BRCA2 Protein; D001483:Base Sequence; D017931:DNA Primers; D005453:Fluorescence; D007700:Kinetics; D015335:Molecular Probes; D009363:Neoplasm Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D013816:Thermodynamics; D014157:Transcription Factors / Q000235:genetics
D024682:BRCA2 Protein; D017931:DNA Primers; D015335:Molecular Probes; D009363:Neoplasm Proteins; D014157:Transcription Factors