Including a second, internal quencher in qPCR probes shortens the distance between 5’ dye and quencher and, in concert with the 3’ quencher, provides greater overall dye quenching, lowering background and increasing signal detection in qPCR experiments (Figure 1A).ZEN and TAO Quenchers can be used for this purpose.
The decrease in background enables the use of more probes in a multiplex qPCR experiment by reducing crosstalk between channels; additionally, multiple probes can be used to test for the presence of a gene in one detector channel (see Figure 1 in the article, Optimizing Multiplex qPCR for Detecting Infectious Disease and Biothreat Agents in the Field).
The additional ZEN™ or TAO™ quencher also increases assay sensitivity, as evidenced by earlier Cq values (Figure 1B). This greater sensitivity can be crucial for detecting targets that are limited due to small sample size or low target expression. In fact, the performance of ZEN Double-Quenched Probes has enabled applications that would otherwise not have been possible .
Figure 1. Increased signal detection and greater assay sensitivity using ZEN™ Double-Quenched Probes. (A) ZEN Double-Quenched Probes (dark blue curves) provide greater dye quenching, producing lower background and, therefore, higher signal intensities than standard single-quenched probes (BHQ® Quenchers; purple curves). (B) ZEN Double-Quenched Probes increase assay sensitivity, as demonstrated by the earlier Cq values observed, compared to standard, single-quenched probes (BHQ Quenchers).
Figure 2 shows that the decrease in background fluorescence is also evident with longer probes. The ability to use longer probes without sacrificing assay performance provides greater flexibility when designing assays; e.g., when interrogating regions of low complexity, it may be necessary to design longer probes to achieve a higher Tm.
Figure 2. ZEN Double-Quenched Probes provide low background even with long probes. 5’ FAM probes of various lengths (40, 35, 30, 25, and 20 nt) with 5 different quenchers: ZEN™ and Iowa Black® FQ (ZEN/IBFQ) Double-Quenched, Black Hole Quencher (BHQ), Eclipse™, Iowa Black FQ (IBFQ), and TAMRA, giving a total of 25 different probe types, were tested for background fluorescence. Cycling was performed using 0.24 μM probe, 50 ng cDNA and Applied Biosystems TaqMan® Gene Expression Master Mix in 6 replicate 10 μL reactions, and standard cycling conditions on the Applied Biosystems 7900HT Fast Real-Time PCR System. Mean background (Rn) measurements were calculated to determine average background fluorescence. Error bars show standard deviation.
Use of ZEN and TAO Double-Quenched Probes provides researchers both increased sensitivity and precision in their qPCR experiments. qPCR probes that include the ZEN or TAO quencher (Double-Quenched Probes) can be ordered online from our Prime- Time® qPCR Probes page.