| In most PCR assays, separate primers
and probes are used to amplify and detect a nucleic acid. An emerging
technique uses a primer and probe linked to each other so that the binding
of the probe to the amplicon is now a unimolecular reaction instead
of a bimolecular one. This oligonucleotide that consists of a probe
and primer linked together is called a Scorpion probe
or Scorpion primer. The advantage of this technique
is that the detection of the amplicon is much faster due to the unimolecular
reaction.
The probe part of a Scorpion is complementary to the extension product
of the primer it is attached to. After the primer has been extended
during the extension step in a PCR cycle, the probe will bind to the
extended part when the complimentary strands are separated in the denaturation
step of the next PCR cycle.
Visual OMP can be used to design different types of Scorpions and to
simulate these together with targets and other oligonucleotides to predict
the thermodynamic behavior and secondary structure of all possible interactions
in the assay.
The types of Scorpion designs include:
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Q-PCR (Quantitative Polymerase Chain Reaction):
Used in quantitative PCR assays to measure amplification. One Scorpion
primer that consist of a primer and probe linked to each other and
one compatible primer to target the other strand of the target are
designed simultaneously.
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ASO (Allele Specific Oligonucleotide)
Scorpion: Used to amplify and discriminate between two
possible alleles of a target oligonucleotide. Two Scorpions, one
for each allele, and one compatible primer to target the other strand
of the target of both alleles are designed simultaneously. The primer
part of both Scorpions are identical; the probe parts differ so
that one will only bind to one of the alleles and the other only
to the other allele. For example, if a SNP (single nucleotide polymorphism)
is being discriminated, the probes will only differ by one nucleotide.
For ASO Scorpions the variation that is being targeted needs to
lie in the region of the target in between the binding sites of
the primers.
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ARMS (Amplification Refractory Mutation
System) Scorpion: Like an ASO Scorpion, ARMS Scorpions
are used to amplify and discriminate between two possible alleles
of a target oligonucleotide. Again, two Scorpions, one for each
allele, and one compatible primer to target the other strand of
both alleles are being designed. The difference is that the probe
parts of both Scorpions are identical; the primer parts of the Scorpions
are used to discriminate between the two alleles.
For all Scorpion designs in Visual OMP it is possible
to choose between the open and closed Scorpion format. Closed means
that the probe part of the Scorpion will be designed to have two stems
at each end that are complimentary to each other so that it will be
in a beacon-like (link) secondary structure when it is not yet hybridized
to the primers’ extension product. This way a fluor and quencher that
are attached to the 5’ and 3’ ends of the probe are in close proximity
when the Scorpion is free in solution and no fluorescence will be detectable.
When the Scorpion unfolds as the probe binds to the extended primer
the fluor and quencher will be separated and fluorescence can be detected
as to quantify the amount of PCR product.
In the open format, the probe part of the Scorpion does not have a specific
secondary structure in the unhybridized form and contains a fluor. A
separate quencher oligonucleotide is designed simultaneously. This quencher
will bind to the probe part of the Scorpion when the Scorpion is not
bound to its intended target as to prevent fluorescence. As the Scorpion
binds to the target the quencher and probe will be separated from each
other when the probe hybridizes to the extension product of the primer.
Now fluorescence can be detected and used to quantify the amount of
PCR product.
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