The key to probe design lies in the selection of fluorescent and quencher moieties:

a. Choose appropriate fluorescent moieties based on the fluorescence quantitative PCR instrument.

b. Select suitable quencher moieties to pair with the chosen fluorescent moieties.

Taqman probes include four selectable quencher moieties: BHQ series, MGB, TAMRA, and Eclipse.

BHQ Probes

The BHQ series includes BHQ-0 (430-520 nm), BHQ-1 (480-580 nm), BHQ-2 (550-650 nm), and BHQ-3 (620-730 nm). These four BHQ quencher groups together cover the absorption spectrum from 430 nm to near-infrared, and they can quench any fluorescent reporter group within this range. They are suitable for multiplex qPCR.

BHQ quencher groups act like black holes, as they do not emit fluorescence themselves and can absorb all fluorescent signals emitted by reporter groups within a certain range. They address the issue of light contamination in qPCR experimental systems by reducing background signals and improving the signal-to-noise ratio of the fluorescence signal.

MGB Probe

MGB probes are dual-labeled probes that are optimized and improved based on TaqMan probes. They contain a minor groove binder (MGB) at the 3' end, which can insert into the minor groove of DNA, thereby increasing the probe's melting temperature (typically by around 10°C) and stabilizing the probe-target hybridization. This means that TaqMan MGB probes can be much shorter than traditional probes, providing better sequence discrimination ability and flexibility to target a wider range of sequences.

The MGB is linked to a non-fluorescent quencher (NFQ), which quenches the signal generated by the fluorescent reporter group at the other end. This avoids the background signal caused by traditional quenchers, resulting in lower fluorescent background and improved detection sensitivity.

TAMRA Probe

TAMRA is a fluorescent dye that can be used as a reporter group for dual-labeled probes and molecular beacons, as well as an internal quencher group (TAMRA-dT). The fluorescence characteristics of TAMRA result in spectral overlap between the quencher group and the reporter group, leading to increased background signal interference. TAMRA probes are typically longer (30-40 nt) compared to other modified probes. However, the use of TAMRA probes is not recommended in multiplex qPCR processes.

Eclipse Probe

Eclipse is a dark quencher, a non-fluorescent dye that does not produce fluorescence itself. Compared to TAMRA probes, Eclipse has lower background signal and a wider absorption range, making it suitable for multiplex qPCR reactions.