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.
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)