Duplex Scorpion primers in SNP analysis and FRET applications

Scorpions are fluorogenic PCR primers with a probe element attached at the 5'-end via a PCR stopper. They are used in real-time amplicon-specific dete ction of PCR products in homogeneous solution. Two different formats are po ssible, the 'stem-loop' format and the 'duplex' format. In both cases the p robing mechanism is intramolecular. We have shown that duplex Scorpions are efficient probes in real-time PCR. They give a greater fluorescent signal than stem-loop Scorpions due to the vastly increased separation between flu orophore and quencher in the active form. We have demonstrated their use in allelic discrimination at the W1282X locus of the ABCC7 gene and shown tha t they can be used in assays where fluorescence resonance energy transfer i s required.