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References >> Scorpion
Scorpion Primers & ProbesScorpion technique was developed by Dr. David Whitcombe of DxS Ltd. Scorpion primers are bi-functional molecules in which a primer is covalently linked to the probe. The molecules also contain a fluorophore and a quencher. In the absence of the target, the quencher nearly absorbs the fluorescence emitted by the fluorophore. During the Scorpion PCR reaction, in the presence of the target, the fluorophore and the quencher separate which leads to an increase in the fluorescence emitted. The fluorescence can be detected and measured in the reaction tube.
The Scorpion primer carries a Scorpion probe element at the 5' end. The probe is a self-complementary stem sequence with a fluorophore at one end and a quencher at the other. The Scorpion primer sequence is modified at the 5'end. It contains a PCR blocker at the start of the hairpin loop (Usually HEG monomers are added as blocking agent). In the initial PCR cycles, the primer hybridizes to the target and extension occurs due to the action of polymerase. Scorpion primers can be used to examine and identify point mutations by using multiple probes. Each probe can be tagged with a different fluorophore to produce different colors.
In Scorpion primers, the probe
is physically coupled to the primer
which means that the reaction leading
to signal generation is a unimolecular
one. This is in contrast to the bi-molecular
collisions required by other technologies
such as TaqMan® or Molecular Beacons.
After one cycle of PCR extension completes,
the newly synthesized target region
will be attached to the same strand
as the probe. Following the second cycle
of denaturation and annealing, the probe
and the target hybridize. The denaturation
of the hairpin loop requires less energy
than the new DNA duplex produced. Consequently,
the hairpin sequence hybridizes to a
part of the newly produced PCR product.
This results in the separation of the
fluorophore from the quencher and causes
emission.
Applications of Scorpion Probes
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1. Amplicon Length: The primer pair should be designed to give an amplicon of approximately 100-200 bp.
2. Secondary Structures: The designed primers should be tested for hairpins and secondary structures. Ideally the primers should have as little secondary structure as possible.
3. Tm Criteria: The Tm's of the two primers should be similar. Also, the stem Tm should be 5-10ºC higher than the probe Tm.
4. Complementary Probe: The scorpion® should be written as the reverse complement of the target.
5. Length Criteria: Probe sequences should ideally be about 17-27 bases Also, the probe target should be 11 bases or less from the 3' end of the scorpion®.
6. Probe Stem: The stem sequence can be of 6 to 7 bases, mostly Cs and Gs, avoiding motifs. The 5' stem sequence should begin with a C as G may quench the FAM.
7. Primer Probe Hybridization: There is always the possibility of the primer hybridizing to the probe element, this will lead to linearization of the probe in an amplification-independent manner causing significant, target-independent fluorescence.