Quenchers
Dabcyl Labeling
Dabcyl, because of its light absorbance properties and lack of residual fluorescence, has most commonly been used as a quencher in diagnostic probes such as Molecular Beacons™.4 A Molecular Beacon™ is essentially a hybridisation probe consisting of a fluorophore, a quencher and a defined oligonucleotide sequence complementary to that of the target nucleic acid. In the inactive state, when the probe is not hybridised to its target sequence, the fluorescence energy of the fluorophore is transferred to the quencher by a process of static quenching. For light energy transfer to take place efficiently, both fluorophore and quencher have to be in close proximity. This requirement is accounted for in the design of Molecular Beacons™. Hybridisation of the Molecular Beacon™ probe to its target sequence results in a breakage of the Hbonds keeping fluorophore and quencher juxtaposed, allowing them to separate, resulting in fluorescence.
Dabcyl can easily be introduced at many positions at the 5’-end, at the 3’-end, or internally by substituting any suitable dT residue with Dabcyl-dT. Dabcyl’s absorption properties limit the range of dyes it can quench, which is restricted to those emitting at 400-550nm (absorption maximum, 471nm). In the design of Molecular Beacons™, the fluorophore and Dabcyl are brought close enough to allow a slightly broader spectrum of dyes to be quenched, thereby increasing the versatility of the Dabcyl molecule
5-Dabcyl
internal dabcyl (5', internal)
TAMRA (Rhodamine) Labelling
The light-absorbing properties of TAMRA, and spectral overlap with several commonly used fluorophores - including FAM, HEX, TET and JOE, make it useful as a quencher for the design of dual-labelled probes. The usefulness of TAMRA is, however, limited because of its broad emission spectrum, which reduces its capabilities in multiplexing. Its intrinsic fluorescence contributes to the background signal, potentially reducing the sensitivity of assays based on TAMRA. Despite these limitations, TAMRA has been used extensively in the design of probe-based assays.
TAMRA (3')
Black Hole Quenchers™ (BHQs)
The demands of modern genomics research and diagnostics, which are typically centred around an ever increasing need for greater assay sensitivity, has led to the development of a series of new dye quenchers. Some of the best known of these are the Black Hole Quenchers™ (BHQs) that have been specifically optimised for FRET-based quenching.
Because of the broad spectral overlap covered by each dye, the efficiency of quenching is increased, when compared to molecules like Dabcyl. This in turn means that BHQs as a set of dyes provide access to a much larger range of wavelengths
for reporting purposes, covering visible into near IR regions of the spectrum (480-730nm). Coupling this with the fact that these molecules have no residual background fluorescence (they are true dark quenchers) means that a greater choice of dyes is available for use in multiplexing, leading to a wider variety of probe formulations
BHQ-1
is typically used to quench in the range 480-580nm and can be used in conjunction with the commonly used fluorophores; e.g. FAM, TET, JOE, Yakima Yellow and HEX.
BHQ-2
is used to quench in the range 559-670nm and is most effective in quenching fluorophores such as TAMRA, ROX, Cy3, Cy3.5, Cy5
and LC Red 640.
