Quenchers Design and Protocols

Quenchers Design / Protocol

The development of a wide variety of fluorescent dyes and quencher molecules for use as detection systems for nucleic acid hybridization probes has permitted the development of several different real-time PCR assay systems that utilize dye-quencher hybridization probes that produce a fluorescent signal only when they bind to their target. However, in order to properly design such an assay for a particular target(s) of interest, it is important to carefully select the dye and quencher of hybridization probes, based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay.

1) Choose fluorescent dyes that can be properly excited and detected by the excitation source and the optics of the spectrophotometric thermal cycler you plan to use in your experiments. Laser-based excitation sources typically provide optimal excitation within a narrow wavelength range (e.g., 500-540 nm for argon ion lasers), while white light sources (e.g., tungsten-halogen lamp) and light-emitting diodes in combination with excitation and emission filters can excite fluorophores within the entire visible range (400-700 nm).

2) For detection of one target with one hybridization probe, FAM, HEX, or TET are recommended for the dye, since they can be detected on all spectrophotometric thermal cyclers, and phosphoramidites are available for all three (which makes synthesis of the probe straightforward and cost-effective).

3) For multiplexed assays, where at least two different hybridization probes are necessary, dyes with well-separated absorption and emission wavelength should be chosen.

4) For FRET hybridization probes, fluorescent dye-quencher pairs with a high degree of spectral overlap should be chosen. See Quencher Selection table to select appropriate quencher.

5) For contact quenching-based hybridization probes (e.g., molecular beacons), ANY non-fluorescent quencher should work well.

Dye & Quencher Selection Table

Dye & Quencher Selection Table

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.