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Detection of PCR products using self-probing amplicons and fluorescence

Abstract

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.

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Figure 1: Detection of PCR products with self-probing amplicons.
Figure 2: Amplicon detection and allelic specificity.
Figure 3: Comparison between bimolecular probing and the unimolecular probing in real-time PCR.
Figure 4: Fluorescence accumulation patterns for three fluorescence signaling methods.
Figure 5: Two possible formats for combining two allelic reactions in a single tube.
Figure 6: Single-tube genotyping.

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Acknowledgements

We thank Dr. Neil Gibson for helpful discussions, Kay Callaghan and Kemal Haque for sharing of unpublished K-ras data, and Dr. Julian Sorrell for information concerning IL-1A polymorphisms. The methods described in this paper form part of worldwide patent applications. ARMS is the subject of European patent number 0332435 (ZENECA Ltd.) and corresponding US patent #5595890. The use of nonamplifiable tails and blocking monomers are the subjects of European patent 0416817 (ZENECA Ltd.) and corresponding US patent #5525494.

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Correspondence to David Whitcombe.

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Whitcombe, D., Theaker, J., Guy, S. et al. Detection of PCR products using self-probing amplicons and fluorescence . Nat Biotechnol 17, 804–807 (1999). https://doi.org/10.1038/11751

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