Black Hole Quencher (BHQ) dyes

A reporter (such as FAM) and a quencher (such as BHQ-1 label) can join to create a new, non-fluorescent species known as an intramolecular dimer. The biomolecule linking the reporter and quencher acts as a tether and enables intramolecular association between the reporter and quencher.

The reporter and quencher are placed at specific sites in an oligonucleotide so that a change in their distance will produce a maximal change in detectable fluorescence, signalling the event being monitored (often hybridization or nuclease activity). The oligonucleotide acts as a flexible tether linking the fluorescent reporter and quencher.

Black Hole Quenchers (BHQ) are a family of dark quencher dyes with excellent spectral overlap with all common reporter dyes for efficient FRET quenching. BHQ labels have become the industry standard for quenchers in dual-labeled hydrolysis probes and many other molecular probe formats.

Black Hole Quencher dyes are true "dark" quenchers with no fluorescent signal, improving signal to noise ratios. Unlike other quenchers, they provide accurate results without any residual background signal through a combination of FRET and static quenching. Their complete quenching simplifies design, implementation and interpretation of qPCR assays.

BHQ dyes are true dark quenchers with no native emission due to their polyaromatic-azo backbone. Substituting electron-donating and withdrawing groups on the aromatic rings produces a complete series of quenchers with broad absorption curves that span the visible spectrum.

BHQ dyes work through a combination of FRET and static or contact quenching to enable researchers to avoid the residual background signal common to fluorescing quenchers such as TAMRA, or low signal to noise ratio. These quenchers can be paired with all common reporter dyes to construct efficiently quenched qPCR probes for multiplexing assays. In addition to quenching by FRET, BHQ dyes have also been shown to efficiently quench fluorescence through static or contact quenching via formation of an intramolecular ground state complex with the reporter dye.

TAMRA dye is an effective quencher for fluorophores with emission maxima less than 560 nm. Dyes with longer wavelength emissions will not be effectively quenched by TAMRA. In addition, TAMRA has its own fluorescence which complicates data analysis due to crosstalk between the channels. In contrast, Black Hole Quencher™ dyes are true "dark" quenchers with no fluorescent signal, therefore using them simplifies design, implementation and interpretation of qPCR assays.

Furthermore, Black Hole Quencher dyes have broad absorption spanning 480-580 nm (BHQ-1), 559-670 nm (BHQ-2) and 620-730 nm (BHQ-3), to enable use of a large range of spectrally distinct reporter dyes in multiplexed assay designs. With some dye pairings, FRET quenching is supplemented by the static quenching mechanism.

Specifically, hydrophobic and electrostatic interactions facilitate the association of BHQ dyes with certain reporters to form an intramolecular dimer, for enhanced quenching and improved signal to noise ratios. Thus, BHQ dyes may quench some fluorophores whose emission spectrum is beyond the limits of BHQ absorption.