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Upping the Sensitivity and Accuracy of qPCR

Double-Quenched Probes Designed to Be Stable at High Temperatures and Minimize Fluorescence

• Mark Behlke, M.D., Ph.D.
• ,
• Scott Rose, Ph.D.
• ,
• Jaime Sabel

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Double-Quenched Probes

Figure 1. (A) The incorporation of an internal ZEN quencher decreases the distance between quencher and dye to only 9 base pairs. (B) The 5’ to 3’ exonuclease activity of the polymerase degrades the probe, physically separating the quenchers from the fluorophore, which results in measurable fluorescence. Click Image To Enlarge +

Figure 1. (A) The incorporation of an internal ZEN quencher decreases the distance between quencher and dye to only 9 base pairs. (B) The 5’ to 3’ exonuclease activity of the polymerase degrades the probe, physically separating the quenchers from the fluorophore, which results in measurable fluorescence.

A number of factors must be considered when designing a self-quenching probe, including ease of synthesis, fluorophore and quencher compatibility, duplex stability, and probe specificity. The newly developed double-quenched probe incorporates a ZEN™ quencher.

The quencher is placed internally at a fixed distance from the 5´-fluorophore, regardless of probe length, in addition to the standard 3´ quencher (Figure 1). The quencher is inserted into the oligo using a nontraditional, performance-enhancing attachment method that requires a special linker. This, in combination with the unique chemical structure of the ZEN quencher, stabilizes duplex formation (increasing T_m) instead of disrupting duplex formation (decreasing T_m) as normally occurs when nonbase modifying groups interrupt a DNA sequence. Overall, this leads to increased functional performance of the probe.

During the annealing phase of each PCR cycle, the primers and double-quenched probe both bind complementary sections of the DNA. During the elongation phase, polymerization of the new DNA strand is initiated from the primers. Once the polymerase reaches the bound probe, its 5´ to 3´ exonuclease activity degrades the probe, thereby physically separating the quencher from the fluorophore (Figure 1). As a result, fluorescence can be measured and will increase in real-time with the exponential increase in PCR product.

Due to the smaller R_FQ achieved with double-quenched probes, background fluorescence is significantly reduced. As such, the initial fluorescence signal measured is much lower, which makes any change in fluorescence easy to detect and functionally increases assay sensitivity.

Figure 2. Probes with five different quenchers and five different base pair lengths (20, 25, 30, 35, and 40) were tested for a total of 25 different probe types. Six replicates of each probe type were mixed with 10 ng/µL cDNA and a gene-expression master mix and run under standard cycling conditions. Mean background (Rn) measurements were calculated to determine the average background fluorescent signal. Click Image To Enlarge +

Figure 2. Probes with five different quenchers and five different base pair lengths (20, 25, 30, 35, and 40) were tested for a total of 25 different probe types. Six replicates of each probe type were mixed with 10 ng/µL cDNA and a gene-expression master mix and run under standard cycling conditions. Mean background (Rn) measurements were calculated to determine the average background fluorescent signal.

In addition, this design enables longer probes to be used with no loss of sensitivity or quality of quenching because the internal quencher is always the same distance from the fluorophore, regardless of probe length. This is especially useful for AT-rich sequences, where long probes may be needed. In order to test the effectiveness of this double-quencher design, several qPCR reactions were run on a real-time PCR instrument, using a variety of different probes.

Since it is such an important issue to ensure that signal-to-noise ratio remains high, background fluorescence needs to be maintained at a minimal level. Significantly lower background fluorescence is observed when using the new double-quenching probe in comparison to various other dual-labeled probes (Figure 2).

Figure 3 (A and B). 5’ FAM probes with five different quenchers: Each probe was synthesized to target the ACTB locus and all were run in triplicate with the same primer products and 0.5 ng of cDNA. Click Image To Enlarge +

Figure 3 (A and B). 5’ FAM probes with five different quenchers: Each probe was synthesized to target the ACTB locus and all were run in triplicate with the same primer products and 0.5 ng of cDNA.

Precision and accuracy are vital in any experimental set-up. Incorporating double-quenched probes produces extremely accurate (Figure 3) and precise data in comparison to a variety of traditional probes.