FLUOROPHORES

Traditionally, FAM-TAMRA is one of the most frequently used pairs for TaqMan probes in which FAM acts as the fluorophore and TAMRA as the quencher. Other quenchers can also be used, especially dark quenchers or black hole quenchers (BHQs) that capture energy from an excited reporter molecule without subsequent emission of light, i.e. they do not fluoresce.

Probes made by using dark quenchers tend to be more sensitive in quantitative detection systems. This is primarily due to lower background fluorescence and a better signal-to-noise ratio than probes that contain fluorescent quenchers. Moreover, dark quenchers enable the use of a wider range of reporter dyes, which expands the options available for multiplexed or genotyping assays.

Some fluorophores can be quenched by more than one quencher. In any case the absorption spectrum of the quencher needs to have a good overlap with the emission spectrum of the fluorophore to achieve optimal quenching. Quenchers have a quenching capacity throughout their absorption spectrum, but the performance is best close to the absorption maximum.

For multiplex PCR we recommend the following 5’ fluorophore 3’ black hole quencher combinations (are suitable for most qPCR thermo cyclers using TaqMan probes):

• Channel 1: FAM-MGBQ530
• Channel 2: HEX/JOE/Yakima Yellow*- MGBQ530 (*equivalent to VIC)
• Channel 3: ROX**-BHQ-2 (**equivalent to Texas Red)/ATTO550***-BHQ-2 (***equivalent to NED)
• Channel 4: Cy5-BHQ-2
• Channel 5: Dy681-BHQ2

Recommended final concentrations for standard experiment.