What is FRET state? - (Mar/03/2008 )
I'm preparing for a seminar that is not related with my research topic. The topic is single-molecule FRET. I am reading literatures and kind of stuck in understanding an important concept: what is FRET state?
the background - you probably know this by now - FRET is "Forster" or "Fluorescent resonance energy transfer", a physical condition which one molecule with weak fluorescence (or emitted energy) absorbs another's stronger fluorescent, etc. energy, and thus no fluorescence or energy is detectable outside the couple's most immediate space (it's rather romantic, i think...). TaqMan-style pcr utilizes this technology - a short oligo (the probe, which is flanked up- and down- stream by forward and reverse primers) is labelled at the 5' end with a strong fluorophore (called "the reporter"), and also labeled at the 3' end with a weak fluorophore ("the quencher"), or some other kind of absorbent compound. Because of their proximity, neither will 'shine' - one basically absorbs the other's energy. As polymerase approaches the probe from 5' direction, it kicks off the reporter from the probe, releasing it into the pcr soup. As the reporter gains distance from the quencher (perhaps it's not so romantic after all, poor smothered reporter...), its native fluorescence is no longer absorbed by the quencher, and the energy is detectable (and detected) by the real-time pcr machine, indicating a postive amplification. Cool.
But that's all i really know. I would guess the FRET state is simply the condition in which the two molecules are energetically 'coupling'... in the case of TaqMan, after probe binding and prior to polymerase cleavage.
Sorry if i wonked off topic, i hope that was helpful.
nice answer from masmith; I agree with the explanation of FRET state, however, a context-related condition may be meant...