Automatic genotyping assay using ScorpionTM primers, MBSR® thermocyclers and Fluoroskan Ascent®
Author: Dr Antony Halsall, DxS Ltd*, Manchester, UK
Introduction
Scorpions is a class leading PCR detection technology with significant benefits over comparable approaches. These include stronger signals, lower backgrounds, faster reactions, simplified design and improved Single Nucleotide Polymorphism tests.
Scorpions are bi-functional molecules containing a PCR primer element covalently linked to a probe element. The molecules also contain a fluorophore that can interact with a quencher to reduce fluorescence. When the molecules are used in a PCR reaction the fluorophore and the quencher are separated which leads to an increase in fluorescence from the reaction tube.
The benefits of Scorpions derive from the fact that the probe element is physically coupled to the primer element - this means that the reaction leading to signal generation is a uni-molecular rearrangement. The benefits of a uni-molecular rearrangement are significant - as the reaction is effectively instantaneous it occurs prior to any competing or side reactions such as target amplicon re-annealing or inappropriate target folding. This leads to stronger signals, more reliable probe design, shorter reaction times and better discrimination (Whitcombe, et al. 1999).
Scorpions primers can be used to detect SNPs by making the probe or primer sequences allele specific (Thelwell, et al. 2000). Allelic variants of a SNP can be detected in a single reaction by labelling the different versions of the probe with different fluorophores, typically FAM and ROX, though Scorpions are compatible with any fluorescent dye.