Quenchers Applications

Quenchers Applications

Dark quenchers are typically used in fluorescent dye-quencher probes requiring suppression of the dye’s fluorescence under one set of circumstances, but not under another, such as in the case of TaqMan and Molecular Beacons probes. Common examples of dark quenchers are Dabcyl and the three Black Hole Quenchers (BHQ). Taken together, the absorption spectra of these four dark quenchers span the entire visible range, which provides a researcher with broad flexibility in choice of fluorescent dye, along with the ability to search a sample for multiple targets in a multiplex quantitative PCR format. Fluorescent quenchers, such as TAMRA, are typically used in fluorescence resonance energy transfer (FRET)-based applications. FRET probes contain a donor (fluorescent dye)-acceptor (fluorescent quencher) pair in close proximity. After absorbance of light by the donor moiety, the donor’s fluorescence emission energy is absorbed (quenched) by the acceptor moiety, and subsequently emitted at the acceptor’s emission wavelength. The result is a final fluorescence emission at a substantially longer wavelength than would be expected if only the donor moiety were present (4). FRET probes thus are useful in cases where a substantial shift in final emission wavelength is desirable. This type of FRET-based system is typically used to determine intra- and inter-molecular distances at very high resolution (1-10 nm) (5). For example, FRET oligo probes have been used to measure the dynamic changes in intermolecular distances between tRNAs bound at the A and P sites of ribosomes during mRNA translation (6). FRET oligo primers have also been used to obtain direct evidence of strand slippage during in vitro synthesis of poly(dG)-poly(dC) duplexes by the Kleno exo- fragment of DNA polymerase I (7).

References

(1) Lakowicz, J.R. Quenching of Fluorescence. in Principles of Fluorescence Spectroscopy, 3rd Edition (2006), Springer-Verlag, Berlin, 278-330.
(2) Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. (1995), 4: 1-6.
(3) Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996), 14: 303-308.
(4) Lakowicz, J.R. Quenching of Fluorescence. in Principles of Fluorescence Spectroscopy, 3rd Edition (2006), Springer-Verlag, Berlin, 331-352.
(5) Stryer, L., Haugland, R.P. Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA (1967) 58: 719-726.
(6) Munro, J.B., Altman, R.B., O’Connor, N., Blanchard, S.C. Molecular Cell (2007), 25: 505-517.
(7) Kotlyar, A.B., Borovok, N., Molotsky, T., Fadeev, L., Gozin, M. In vitro synthesis of uniform poly(dG)-poly(dC) by Klenow exo(-) fragment of polymerase I. Nucleic Acids Res. (2005), 33: 525-535.