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.
- 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 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.
- Enhanced specificity: The stem-loop conformation confers high specificity, making Scorpions primers ideal for SNP/mismatch discrimination.
- High signal-to-noise: Scorpions primers offer superior fluorophore quenching due to the proximity and the direct energy transfer between reporter and quencher while in the closed hairpin conformation.
- Melt curve analysis suitability: Because Scorpions primers generate fluorescence under non-hydrolytic conditions by target hybridisation, post-PCR melt curve analysis can be performed.
- Rapid hybridisation: The proximity of the probe region and the target sequence kinetically favours formation of the probe-template hybrid over template duplex re-annealing. The unimolecular event enables rapid signal generation during hybridization.
Select the best dye for your application
We offer Scorpions primers labelled with a wide selection of dyes, enabling you to select options based on instrumentation and assay design. Quencher options for this probe type include Black Hole Quenchers, BHQ-1 and BHQ-2.
5' fluorescent dye
|CAL Fluor Gold 540||522||544||BHQ-1||HEG|
|CAL Fluor Orange 560||538||559||BHQ-1||HEG|
|CAL Fluor Red 590||569||591||BHQ-2||HEG|
|CAL Fluor Red 610||590||610||BHQ-2||HEG|
|CAL Fluor Red 635||618||637||BHQ-2||HEG|
Use the spectral overlay tool to view compatible dyes for your instrument. Learn more