When to use an internal Black Hole Quencher (BHQ) label in a fluorescence-quenched probe for real-time qPCR.
When the sequence length of your probe is 30 bases or shorter, we recommend selecting a conventional BHQ label for the 3' end of your probe. We have noticed, however, that probe lengths longer than 30 bases exhibit high background noise and are frequently nonfunctional. They are too long for efficient FRET quenching, a mechanism that is highly dependent upon the distance between the fluorophore and quencher. A shorter average distance between fluorophore and quencher is optimal for a FRET quenching environment, which is common in real-time qPCR experiments.
To resolve the problematic issues that could potentially arise when designing a longer probe, we recommend using an internal BHQ label, and putting a Spacer 3 (C3) modification at the 3' end to block polymerase extension. Without this Spacer 3 modification, the probe would instead behave as a primer and produce non-specific amplicons, which may be observed as a false positive. Placing your quencher internally can significantly reduce background noise because the quencher is positioned in closer proximity to the 5' fluorophore.
Biosearch Technologies offers T-BHQ-1, an internal BHQ dye attached to a thymine (T) base. The internal BHQ label should be positioned no closer than 10 bases from the 5’ end to minimize interference. Visit our dual-labeled probes product page to order probes internally labeled with a BHQ dye. Simply select FAM from the 5’ Fluorophore drop down menu and an Internal Quencher drop down menu will appear with the option to select T-BHQ-1.