PCR and qPCR
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Increase sensitivity and precision in your qPCR experiments

ZEN™ and TAO™ Double-Quenched Probes improve qPCR data

While traditional qPCR probes have approximately 20–30 bases between the fluorophore and the quencher, including an internal ZEN™ or TAO™ quencher decreases that length to only 9 bases (Figure 1). This shortened distance, particularly when combined with a traditional 3’ end quencher, provides more thorough quenching with much lower background. Further, it enables use of longer probes, which are especially beneficial for qPCR assays designed in AT-rich target regions.

Figure 1. An internal quencher positioned to provide more thorough quenching, lowering background.

In addition to the significantly decreased background, assay data generated using ZEN or TAO Double-Quenched Probes also have consistently reduced Cq values and improved precision when compared to traditional probes (Figure 2). Use Double-Quenched Probes to experience both increased sensitivity and precision in your qPCR experiments.

Figure 2. Double-Quenched Probes increase assay sensitivity. ZEN™ Double-Quenched Probes (dark blue curves) provide greater dye quenching, producing lower background and, therefore, higher signal intensities than standard single-quenched probes (purple curves = BHQ® Probes).

ZEN Double-Quenched Probes

ZEN Double-Quenched Probes contain a 5' fluorophore; a 3' IBFQ quencher; and a proprietary, internal ZEN quencher. The most commonly supplied 5' fluorophores include FAM, Hex™, TET™, MAX™, and JOE. For more information download the PrimeTime® Custom qPCR Probes Flyer.

TAO Double-Quenched Probes

TAO Double-Quenched Probes are available with a 5' Cy® 5 fluorophore; 3' IBRQ quencher; and a proprietary, internal TAO quencher.

Design your Double-Quenched Probes now!

What your colleagues are saying about Double-Quenched Probes:

“We were originally using MGB probes (Applied Biosystems) in our qPCR assays, but recently switched to ZEN Double-Quenched Probes. ZEN Probes are a really good fit for our methylation biomarker work. ZEN Double-Quenched Probes can be up to 40 nt long and still remain quenched due to the shorter proximity of the ZEN quencher to the reporter dye. Additionally, the ZEN moiety increases probe Tm. This means that they actually work really well for this type of application.”

– Dr Antoinette Perry, Senior Research Fellow, Institute of Molecular Medicine, Trinity College (Dublin, Ireland)

“In addition to providing us with the ZEN Double-Quenched Probes to help resolve our background issues, IDT has been invaluable, both from a quality and turnaround time perspective. We frequently make use of being able to order primers and receiving them the next day.”

– Rolf Rauh, Scientist at Tetracore (Rockville, MD, USA)

Product focus: Assays, probes, and controls for qPCR and PCR

PrimeTime® qPCR Assays

- 5′ nuclease, probe-based assays—the gold standard for quantitative gene expression studies

- Primer-based assays—designed for intercalating dye experiments

Create custom assays that are designed using our proprietary bioinformatics algorithms for any target and to your specific parameters. Alternatively, select one of our predesigned assays for human, mouse, and rat mRNA targets that are supported by our bioinformatics algorithms and up-to-date sequence information.

Learn more at www.idtdna.com/PrimeTime.

For assistance with assay design, contact our scientific application specialists at applicationsupport@idtdna.com.

Double-Quenched Probes

IDT also makes  TAO™ Double-Quenched Probes available in addition to ZEN™ Double-Quenched Probes. Both have a 5′ fluorophore, the internal quencher (ZEN or TAO quencher), and Iowa Black FQ as the 3′ quencher. These probes provide consistently earlier Cq values and improved precision when compared to traditional, single-quenched qPCR probes.

Learn more at www.idtdna.com/qPCRprobes.

gBlocks® Gene Fragments

gBlocks Gene Fragments are double-stranded, 125–2000 bp DNA molecules. They are ideal for use as qPCR controls and standards, as well as for gene construction and editing applications. These affordable gene fragments are sequence-verified, ship in a few working days, and save laboratory time.

Learn more at www.idtdna.com/gBlocks.

Additional reading

Learn how use of ZEN™ and TAO™ Double-Quenched Probes are helping researchers detect disease and monitor treatment effectiveness:

Zika virus: Advances in disease modeling and detection—IDT is supporting global research aimed at reducing the widespread effects of Zika. Learn about the virus, and read a summary of the latest developments employing ZEN Double-Quenched Probes and IDT PrimeTime® qPCR Assays and gBlocks® Gene Fragments.

Increasing ddPCR performance in low target HIV assays—ZEN Double-Quenched qPCR Probes work well in ddPCR, providing Dr Matthew Strain’s lab additional sensitivity through lower background in experiments with low copy number samples, where individual droplets matter. The Strain lab has published open-access protocols for their HIV assays that include these updated ZEN probe designs at www.bio-protocol.org.

qPCR assays for optimized viral detection in clinical samples—ZEN™ Double-Quenched Probes were used in a unique qPCR experiment to help rapidly and accurately detect the highly variable norovirus in clinical samples.

Double-Quenched Probes Increase Sensitivity of qPCR Assay Detecting Viral Load—Use of a ZEN™ Double-Quenched Probe results in a marked decrease in background fluorescence compared to an identical TaqMan® probe containing only a single quencher. The data suggest that such double-quenched probes may be a better approach for other qPCR probe-based assays.

Optimizing Multiplex qPCR for Detecting Infectious Diseases and Biothreat Agents in the Field—Researchers at Tetracore specialize in developing large sets of robust probe-based qPCR assays for use in a multiplex format to detect infectious diseases and bio-terrorism threat agents. Here they discuss the need to: use probe dyes compatible on common PCR instruments, maintain low background with multiple probes, and reformulate assays to address viral mutation; and how ZEN™ Double-Quenched Probes have helped meet these criteria.

Visit the IDT online newsletter, DECODED, for more application overviews, experimental tips, and examples of using IDT products in published research by scientists like yourself.

Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.

© 2015, 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

Predesigned qPCR Assays

Probe-based qPCR assays for quantification of human, mouse, and rat gene expression. Order in plates or tubes.

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