Dark quencher

A quencher is a non-fluorescent dye that absorbs light but does not emit it. It is used in molecular biology in conjunction with regular fluorophores: when they are within range no emissions are detected but when they are separated the flourophore's emission is detected.

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SNP Genotyping Example

An example of its use is in Taqman or invader assay, SNP genotyping methods. for example a hairpin loop with a fluorophore and quencher at the base of the stem is used: a unlabelled SNP specific PCR primer (one of many) with a specific 5' tail binds to the sequence to be probed, the taq polymerase extends the sequence that will have a specific 5' end dependent on the SNP (insensitive to polymorphisms upstream of the SNP in question), in the next run a primer, complementary to that tail, with a haipin loop is extended, in the next run the elongation of the complementary strand will linearise the hairpin separating the fluorophore and quencer. An alternative to using quenchers is by using FRET where the combination of two dyes gives a signal.

Examples of dark quenchers

Dabcyl (dimethylaminoazosulphonic acid) absorbs in the green spectrum and is often used with flourescein. (Dabsyl has a nearly identical absorption but has a sulphonyl choride to form more stable conjugates, instead of a succinimidyl ester). Black Hole Quenchers (Biosearch Technologies) are capable of quenching across the entire visible spectrum. Qxl quenchers from AnaSpec span the full visible spectrum. Iowa black FQ absorbs in the green-yellow part of the spectrum. Iowa black RQ blocks in the orange-red part of the spectrum.

Why are dark quenchers dark?

Dark quenchers are dyes with no native fluorescence. Until the last few years, quenchers have typically been a second fluorescent dye, for example, fluorescein as the reporter and rhodamine as the quencher (FAM/TAM probes). However, quencher fluorescence can increase background noise due to overlap between the quencher and reporter fluorescence spectra. This limitation often necessitates the use of complex data analysis and optical filters. Dark quenchers offer a solution to this problem because they do not occupy an emission bandwidth. Furthermore, dark quenchers enable multiplexing (when two or more reporter-quencher probes are used together).
Fluorescent dyes absorb light, which places the dye in an excited state; the dye returns to the ground state from the excited state by emitting light (fluorescence). In a reporter – quencher system the dye nonradiatively (without light) transfers energy to the quencher. This returns the dye to the ground state and generates the quencher excited state. The quencher then returns to the ground state through emissive decay (fluorescence) or nonradiatively (dark quenching). In nonradiative or dark decay, energy is given off via molecular vibrations (heat). (It should be noted that with the typical μM or less concentration of probe, the heat from radiationless decay is too small to effect the temperature of the solution.)

Quenching Mechanisms

  There are a few distinct mechanisms by which energy can be transferred nonradiatively (without absorption or emission of photons) between two dyes, a donor and an acceptor. Förster resonance energy transfer (FRET or FET) is a dynamic quenching mechanism because energy transfer occurs while the donor is in the excited state. FRET is based on classical dipole-dipole interactions between the transition dipoles of the donor and acceptor and is extremely dependent on the donor – acceptor distance (R), falling off at a rate of 1/R⁶. FRET also depends on the donor-acceptor spectral overlap (see figure below) and the relative orientation of the donor and acceptor transition dipole moments. FRET can typically occur over distances up to 100 Å.

Dexter (also known as exchange or collisional energy transfer) is another dynamic quenching mechanism. Dexter energy transfer is a short-range phenomenon that decreases with e^-R and depends on spatial overlap of donor and quencher molecular orbitals. In most donor fluorophore – quencher acceptor situations, the Förster mechanism is more important than the Dexter mechanism. With both Förster and Dexter energy transfer, the shapes of the absorption and fluorescence spectra of the dyes are unchanged. Exciplex (excited state complex) formation is a third dynamic quenching mechanism.

The remaining energy transfer mechanism is static quenching (also referred to as contact quenching). Static quenching can be a dominant mechanism for some reporter-quencher probes. Unlike dynamic quenching, static quenching occurs when the donor and acceptor molecules are in the ground state. The donor and acceptor molecules bind together to form a ground state complex, an intramolecular dimer with its own unique properties, such as being nonfluorescent and having a unique absorption spectrum. Dye aggregation is often due to hydrophobic effects – the dye molecules stack together to minimize contact with water. Planar aromatic dyes that are matched for association through hydrophobic, electrostatic and steric forces can enhance static quenching. High temperatures and addition of surfactants tend to disrupt ground state complex formation.

References

• J. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum, New York, 1999.
• M.K. Johansson, R.M. Cook, Chem. Eur. J. 2003, 9, 3466-3471.
• Invitrogen Handbook: QSY dyes.