Duplex Scorpion primers in SNP analysis and FRET applications |
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| Authors: | Solinas A Brown L J McKeen C Mellor J M Nicol J Thelwell N Brown T |
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| Affiliation: | Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK. |
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| Abstract: | 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 detection of PCR products in homogeneous solution. Two different formats are possible, the ‘stem–loop’ format and the ‘duplex’ format. In both cases the probing 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 fluorophore and quencher in the active form. We have demonstrated their use in allelic discrimination at the W1282X locus of the ABCC7 gene and shown that they can be used in assays where fluorescence resonance energy transfer is required. |
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