Two quenchers are better than one
Most 5′ hydrolysis probes (dual-labeled) use a terminal fluorophore and a single terminal quencher, with 20−30 bases in between. The level of interaction between the fluorophore and quencher is determined by probe length. IDT ZEN™ PrimeTime® Probes include traditional 5′ fluorophores and 3′ quenchers , but incorporate an additional ZEN quencher into dual-labeled probes between the 9th and 10th bases from the 5′ end. This decreased distance improves quenching, with less background than traditional dye-quencher combinations, resulting in higher signal detection.
Decreased background. A recent study from the New Zealand–based nucleic acid isolation kit manufacturer, ZyGEM , compared the performance of IDT ZEN Double-Quenched Probes with traditional 5′ hydrolysis probes in qPCR assays. Assays differed only in the quenchers used—primer and probe sequences remained constant. All probes used a 5′ FAM™ dye (Applied Biosystems) with either a 3′ TAMRA™ Quencher (Applied Biosystems), a 3′ Black Hole Quencher® (BHQ®, Biosearch Technologies), or an internal ZEN Quencher and 3′ Iowa Black® Fluorescent Quencher (IBFQ). Figure 1 shows the qPCR results for the human BRCA1 gene, and indicates significantly less background for the ZEN Double-Quenched Probe compared to the two dual-labeled probes.
Figure 1. ZEN™ Double-Quenched Probes provide superior background to signal ratio. Human DNA (5 ng) was amplified using Quanta Accustart™ Taq PCR Supermix, two unlabelled primers (0.2 µM each), and 3 traditional dual-labeled probes (see text). Optimal results were obtained with 0.1 µM probe. ZEN Double-Quenched Probes out-perform TAMRA- or BHQ®-quenched probes in both background fluorescence and background to signal ratio. (Figure courtesy of David Saul [ZyGEM].)
Increased sensitivity. ZEN Double-Quenched Probes also show reduced Cq values compared to traditional 5’ hydrolysis probes. A study conducted by IDT compared the average Cq values of a ZEN probe to four other dual-labeled probes. The probe sequences and reporter dyes were identical; only the quenchers differed. The ZEN probe exhibited the lowest average Cq values across each template concentration tested (Figure 2).
Figure 2. ZEN™ Probes exhibit lower average Cq over traditional dual-labeled probes. 5′ FAM probes with 5 different quenchers: ZEN™ Quencher and Iowa Black® FQ (ZEN/IBFQ), Black Hole Quencher® (BHQ), Eclipse® (Epoch Biosciences), Iowa Black FQ (IBFQ), and TAMRA were individually synthesized 10 times per quencher type resulting in 50 unique primer-probe assays. Each reaction used the same primer and probe sequences targeted to a region of the ACTB locus; all were run in triplicate using the same primers and indicated amount of cDNA. Standard curves were the result of a 5-log, 10-fold cDNA dilution starting at 5 ng per reaction. Reactions were run with Applied Biosystems TaqMan® Gene Expression Master Mix under standard cycling conditions on the Applied Biosystems 7900HT Real-Time PCR System.
Improving Your qPCR
The reduced background fluorescence and increased sensitivity of IDT ZEN Probes substantially improves signal detection. In addition, the proximity of the ZEN Quencher to the reporter dye allows probes as long as 40 bases to stay quenched. This increases design flexibility, including possibilities for designing A/T rich probes with practical melting temperatures. These features make ZEN Double-Quenched Probes ideal for improving results from existing assay designs, and making challenging new probe designs possible.
- Tsuei A, Saul D. (2010) Sample normalisation with RNAGEM™ and qPCR using an IDT ZEN PrimeTime probe. ZyGEM (Hamilton, New Zealand), internal report.
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/SNP information.
Learn more at www.idtdna.com/PrimeTime.
For assistance with assay design, contact our scientific application specialists at firstname.lastname@example.org.
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.
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.
Author: Jeremy Pritchard is a Technical Support Representative at IDT.
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