| Scorpion Primers & Probes |
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Introduction to Scorpion®
Primers
Scorpion 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.
Structure of the Scorpion primer
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.
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Scorpion® Primer
Functioning
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. |
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Applications of Scorpion Probes
- SNP
analysis
- Real-time PCR
- Allelic discrimination
- Single tube genotyping assay
Design
Parameters for Scorpion® Primer
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.
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