Days are closing in while heading towards winter. ‘’Bright Probes’’ controlled by efficient Dark Quenchers are in need - license-free, no strings attached – top quality double quenched probes (dqps) by metabion!

Two selection criteria are essential for the length of a linear hydrolysis probe used in 5'-nuclease assays:

  1. Quenching efficiency and
  2. Binding stability.

As such, sequence design is a careful trade-off between these two factors.

In general, and not trying to sell something that isn´t necessary but actually a cost driver, probes up to 25 nts do not require double quenching – dlps are just fine.

In some cases, however, it is unavoidable to extend the probe sequence beyond the favourable 20-30 nt window, i.e. for AT-rich target sequences, to achieve the necessary and acceptable probe Tm for best assay performance. 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 inhibit probe extension during hybridization.

Typically, a DQP's internal Non-Fluorescent Quencher is located between the 9th and 10th nt downstream of the 5'-fluorescent Reporter:

metabion´s standard dqp portfolio offers:

  • 5 yield scales based on delivered quantities tailored to your routine needs;
  • a comprehensive list of license-free Reporter-Quencher combinations;
  • HPLC purification and Mass-Check;

Validate your probe design through our WOP.