Techne PrimeQ - Manual (Page 45)

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fluorescence signal or fluorescence at a longer wavelength which is not detected. The 5’
fluorophore is often called the reporter.
Mode of action of hydrolysis probes:
During the PCR, as the DNA polymerase extends the
upstream (forward) primer, it encounters the bound
probe.
The 5’ to 3’ exonuclease activity of the polymerase
cleaves the probe, releasing the fluorophore into
solution, where it is able to fluoresce.
The probe is blocked at the 3’ end to prevent
extension by the polymerase.
Each cycle of the PCR releases more fluorophore
such that the amount of fluorescence in any given
cycle should be proportional to the amount of specific
product present at any given time.
A particular advantage of the hydrolysis probe technology, aside of the specificity and sensitivity
afforded by all the fluorescent probe chemistries, is that the signal accumulation is irreversible.
Once a probe is cleaved, the quencher is permanently separated from dye and this is reflected in
the signal accordingly.
2.3.3 Molecular Beacons
As with the hydrolysis probe technology, molecular beacons use fluorophore/quencher pairs in
their mode of action. When free in solution, molecular beacons assume a hairpin structure that
brings the end-bound fluorophore and quencher into close proximity thereby quenching the
fluorescent signal. The molecular beacon binds to the amplicon produced during the PCR at a
specific temperature when the beacon-target duplex is thermodynamically more stable than the
hairpin structure. Binding of the beacon to its target disrupts the hairpin, resulting in spatial
separation of the fluorophore from the quencher and allowing it to fluoresce. This increase in
fluorescence is reversible as the beacon will dissociate at a higher temperature and close back to
a hairpin. The transition from hairpin to bound form is repeated each cycle.
Mode of action of molecular beacons:
When free in solution, molecular
beacons assume a hairpin-structure.
Binding of the beacon to its target
disrupts the hairpin, resulting in spatial
separation of the fluorophore from the
quencher and allowing it to fluoresce.
Molecular beacons combine the
specificity of a probe-based chemistry
with the versatility of a reversible binding
reaction.