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 non-functional. 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.

LGC 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.

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