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 Tm) instead of disrupting duplex formation (decreasing Tm) 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 RFQ 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.