Twice dyed probes
Twice Dyed Probes are fluorogenic probes that consist of an oligonucleotide with a 5’-reporter dye and a 3’- quencher dye.
When this probe is intact, the proximity of the reporter dye to the quencher is close enough, so the quencher suppresses the fluorescence of the reporter dye.
During PCR, when the sequence target of interest is present, this probe anneals between the locations of the forward and reverse primers. Now the 5’-3’ nuclease activity of the DNA-polymerase cleaves the Twice Dyed Probe. (See figure 1)
This will only happen when the probe hybridizes to the target, the DNA-polymerase will not cleave the free probes. The cleavage of the Twice dyed Probe results in an increase of fluorescence of the reporter dye. The fluorescence signal is generated only if the target sequence of the probe is amplified by PCR. No signal will be generated due to nonspecific amplification.
Fig 1 to 3: stepwise representation of the cleaving of the fluorophore from the Twice Dyed Probe.
The most frequently used quencher in these probes is TAMRA. With it’s absorption maximum of ~ 555 nm, it efficiently quenches only a limited number of dyes e.g. FAM, JOE, TET and HEX.
Biolegio now offers a new line of quenchers: the Black Hole QuenchersTM.
BHQsTM offer many advantages over TAMRA for a variety of reasons. BHQsTM are dark quenchers and have no native fluorescence while TAMRA is strongly fluorescent and contributes significantly to background fluorescence.
The family of BHQsTM have absorption spectra that cover the visible spectrum. This means that any fluorophore can be matched with a BHQTM for spectral overlap and FRET quenching can be maximized.
"Black Hole Quencher" is a registered trademark of Biosearch Technologies, Inc., all products are licensed and sold under agreement with Biosearch Technologies, Inc.