Duplex Scorpion primers in SNP analysis and FRET applications

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.

Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye

“…Although there are many elegant methods for closedtube genotyping, the oligonucleotides used are often complex, with multiple fluorescent dyes and/or functional groups (26,27 ). For example, in one popular approach, a fluorescent dye, a minor groove binder, and a quencher are all covalently attached to an allele-specific probe (27 ).…”

Overview of Real-Time PCR Principles

“…Scorpions are specific enough for allele discrimination and may be multiplexed easily (Thelwell, Millington et al 2000). The scorpion chemistry has been improved with the creation of duplex scorpions in which the reporter dye/probe and quencher fragment are on separate, complementary molecules (Solinas, Brown et al 2001). The duplex scorpions still bind in a unimolecular event, but because the reporter and quenchers are on separate molecules, they yield greater signal www.intechopen.com intensity because the reporter and quencher can separate completely (Wong and Medrano 2005).…”

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

“…FRET has been successfully used to detect allele-specific products from SBE, 63 ASE, 38,71 DNA hybridization, 13,14,72 DNA ligation 15 and structure specific cleavage. 10 It is the most popular mechanism for homogeneous detection.…”