PCR and qPCR
Support and Educational Content

Double-quenched probes increase qPCR sensitivity and precision

ZEN™ and TAO Double-Quenched Probes

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). 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 [1].



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. 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 provide 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 at Custom qPCR Probes).

References
1. Wilson P, Labonte M, et al. (2011) A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates. Nucleic Acids Res, 1(39):e112.

Author: Hans Packer, PhD, former Scientific Writer, IDT.


Predesigned qPCR Assays

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

Search human, mouse, or rat genes ≫


Related Articles

Designing PCR Primers and Probes

General guidelines for designing primers and probes and for choosing target locations for PCR amplification.

Read more ≫

Steps for a Successful qPCR Experiment

Considerations for 5′ nuclease assay design and experimental setup to help you obtain accurate and consistent results.

Read more ≫

Interpreting Melt Curves: An Indicator, Not a Diagnosis

Examining PCR melt curve data to determine what it can/cannot tell us about resulting PCR amplicons.

Read more ≫

Epigenetic Biomarkers for Prostate Cancer

Scientists use methylation and expression analysis methods to evaluate epigenetic markers for early, noninvasive detection of aggressive prostate cancer. IDT PrimeTime® qPCR Assays, ZEN™ Double-Quenched Probes, and gBlocks® Gene Fragments facilitate this research.

Read more ≫

Optimizing Multiplex qPCR for Detecting Infectious Diseases and Biothreat Agents in the Field—ZEN™ Double-Quenched Probes bring down the background

Tetracore researchers developing large sets of robust probe-based qPCR assays 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.

Read more ≫