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™ and TAO™ PrimeTime® Probes include traditional 5′ fluorophores and 3′ quenchers, but incorporate an additional ZEN or TAO 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.
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].)
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 and TAO probes substantially improves signal detection. In addition, the proximity of the ZEN or TAO 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 adn TAO Double-Quenched Probes ideal for improving results from existing assay designs, and making challenging new probe designs possible.