Enhanced Diagnostic Tools
Fluorescence Resonance Energy Transfer (FRET) is a process where energy from an excited fluorophore is transferred to a neighboring acceptor molecule without the release of light by the fluorophore (quenching). In FRET, the efficiency of this energy transfer process is dependent upon the physical distance between the reporter fluorophore and the quencher and the degree of overlap between reporter emission and quencher absorption spectra. When reporter and quencher are close, quenching is efficient and the oligo is dark. When reporter and quencher are distant, quenching is reduced or eliminated and the oligo is bright. The range over which quenching occurs is unique for each reporter/quencher pair but is commonly between 30 and 70 Å (the distance where quenching is 50% efficient is defined as the Förster radius for that R/Q pair). The quencher (energy acceptor) molecule can be another fluorescent dye such as TAMRATM or a non-fluorescent dark quencher.
How to choose the right quencher?
For the design of fluorescent hybridization probes that utilize FRET, fluorophore-quencher pairs that have sufficient spectral overlap should be chosen. Fluorescent dyes with an emission maximum between 500 and 550 nm, such as 6-FAM, TET and HEX are best quenched by quenchers with absorption maxima between 450 and 550nm, such as DABCYL and BHQ-1TM. Fluorophores with an emission maximum above 550nm, such as Rhodamines (including TAMRA, ROX and Texas Red) and Cy® dyes are best quenched by quenchers with absorption maxima above 550 nm (including BHQ2).
For the design of fluorescent hybridization probes that utilize contact quenching, any non-fluorescent quencher can serve as a good acceptor of energy from the fluorophore. However, it has been shown that CY® dyes are best quenched by the BHQ-2TM and BHQ3TM quenchers.
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