Get Help Sign In

Double-quenched probes increase signal to noise ratios by decreasing background fluorescence

ZEN™ and TAO™ Double-Quenched Probes

Review data demonstrating that ZEN Double-Quenched Probes in qPCR assays provide improved signal to noise ratios compared to single-quenched probes. ZEN and TAO Double-Quenched Probes also make it more feasible to use longer probes due to the lower background fluorescence.

Including a second, internal quencher in qPCR probes decreases the distance between 5’ dye and quencher which, in concert with the 3’ quencher, provides greater overall dye quenching, resulting in decreased background fluorescence and improved signal to noise ratio (Figure 1A). IDT offers ZEN and TAO internal quenchers for use in double quenched probes. 

The additional ZEN or TAO quencher also increases assay efficiency, as illustrated by a decreased number of cycles required to meet the signal threshold (Ct) compared to single-quenched probes (Figure 1B). This can be critical for identifying targets that are limited due to small sample size or low target expression. In fact, the use of ZEN Double‑Quenched Probes rather than standard single-quenched probes enabled applications that were not previously possible due to very high signal to noise ratio requirements when measuring low picomole quantities of dNTPs [1].

Improved qPCR results with ZEN Double-Quenched Probes
Increase signal from ZEN Double-quenched probes. (A) ZEN probes provide greater dye quenching, producing lower background and, therefore, higher signal intensities than standard single-quenched probes (BHQ probes). (B) ZEN probes show earlier observed Cq values compared to BHQ single-quenched probes. Three replicate reactions with each probe type (40 bases long) were run with a gBlock Gene Fragment template (2 x 105 copies) and PrimeTime Gene Expression Master Mix (IDT) on the QuantStudio 7 qPCR instrument (ThermoFisher Scientific).

Figure 2 shows that the decrease in background fluorescence obtained when using double-quenched probes is also evident with longer probes. The ability to use longer probes without sacrificing assay quality provides greater flexibility when designing experiments. For example, when interrogating regions of low complexity, it may be necessary to design longer probes to obtain a higher Tm. Unlike single-quenched probes, ZEN Double‑Quenched Probes exhibit low background fluorescence even when 40 nt long (Figure 2).

low background even with long probes with ZEN Double-Quenched probes
Only ZEN Double-Quenched Probes maintain low background signal with increasing probe length. Probes of either 20 or 40 bases with 4 different quenchers run in 3 replicate reactions with each probe type run with a gBlock Gene Fragment template (2 x 105 copies) and PrimeTime Gene Expression Master Mix (IDT) on the QuantStudio 7 qPCR instrument (ThermoFisher Scientific).

Use of ZEN and TAO Double-Quenched Probes provides researchers with increased precision and design flexibility in their qPCR experiments. qPCR probes that include the ZEN or TAO quencher (Double-Quenched Probes) can be ordered online from our PrimeTime™ qPCR Probes page.

Real-Time PCR handbook


This updated, complete qPCR handbook is full of information to get you on your way to generating great data. Learn how to design your singleplex or multiplex qPCR assay like a pro, how to analyze qPCR data, and more.

Download now


  1. Wilson P, Labonte M, Russel J,  et alA novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates. Nucleic Acids Res, 1(39):e112.


Published Sep 16, 2013
Revised/updated Sep 10, 2023

Black Hole Quencher™ is a trademark of Biosearch Technologies, Inc. Licensed from BTI for RUO.