Scorpions Primers

Scorpions™ primers combine primer and probe into a single bifunctional molecule. They contain a primer sequence at the 3′ end and a hairpin loop structure at the 5′ end. Like molecular beacons, the hairpin brings the reporter and quencher into proximity and the loop contains a sequence complementary to the target.

These primers utilise a unimolecular mechanism that acts faster in solution for instantaneous fluorescence in real-time PCR. Similar to molecular beacons, Scorpions primers do not require enzymatic cleavage of the probe during PCR cycling. These qualities make Scorpions primers valuable tools for rapid, real-time PCR, endpoint PCR, SNP detection and gene quantification.

Scorpions primer structure

These molecules incorporate two distinct structures: a target-specific probe and a target-specific PCR primer.

Probe sequence:

• 5′ fluorophore: The reporter dye emits fluorescence when the probe is linearised and hybridised to the target, separating the dye and quencher.
• 3′ quencher: When the probe is in its hairpin structure, the proximity of the quencher to the reporter prevents fluorescence emission.
• Stem: The stem is a double-stranded region formed by binding the complementary sequences (5-7 nt) at both ends of the probe.
• Loop: The loop is a 18-30 nt sequence that is complementary to the target sequence.

Primer sequence:

• Primer region: A target-specific PCR primer is covalently linked to the 3' end of the probe sequence through a blocking moiety, often a hexaethylene glycol (HEG) modification. This moiety prevents polymerase extension into the probing element, which could generate false positives.

Scorpions primer mechanism

During the first PCR cycle, the primer region hybridises to the complementary DNA template sequence and extends to form a primer extension product. The primer extension product contains the desired probe target sequence. At this stage, the probe region remains in a closed hairpin conformation. The presence of a PCR blocker in the linker sequence prevents the polymerase-mediated duplication of the probe region.

During the second PCR cycle, the target-binding region (loop region) of the probe sequence hybridises to the complementary sequence within the primer extension product. Hybridization forces the hairpin open, separating the fluorophore and quencher, and releasing fluorescence.