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Why Double Quenched Probes?

In accordance with the FRET concept, our DQPs are optimized for real-time quantitative PCR assays, while addressing the need to efficiently mitigate the background noise inherent in probes longer than 25 nts.

Two selection criteria are essential for the length of a linear hydrolysis probe used in 5'-nuclease assays: quenching efficiency and binding stability. As such, sequence design is a careful trade-off between these two factors.

In some cases, to achieve the necessary and acceptable probe Tm for best assay performance, it is necessary to extend the probe sequence beyond the favorable 20-30 nt window, i.e. for AT-rich target sequences. However, as the sequence length increases, the quenching efficiency decreases, leading to enhanced baseline fluorescence and reduced signal-to-noise values for the designed assays.

To overcome this upper probe length limit of 30 nts, it is possible to introduce an internal quencher closer to the fluorescent 5´moiety, while the additional 3'-quencher not only enhances quenching efficiency but also serves as a polymerase inhibitor, thereby avoiding probe extension during hybridization.

Typically, a DQP's internal quencher is located between the 9th and 10th nt downstream of the 5'-fluorescent reporter. This minimizes the distance between the reporter and the quencher and thus the background fluorescence.

R: Reporter/Fluorescent Dye; NFQ: Non-Fluorescent Quencher