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Transitioning your existing hydrolysis probe (HP)-based assays (i.e. TaqMan™ assays) from your current system to Countable PCR is straightforward. It’s possible to use an existing assay with already designed HP and primers directly on the Countable platform; however, we strongly recommend that you include probe additives (PAs) in your existing assays to achieve optimal assay performance on the Countable System.

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Probe compatibility and characteristics

• The Countable PCR platform is compatible with both HPs and Locked Nucleic Acid (LNA®) probes.
  • Most HPs are dual-quenched and feature a 5’ fluorophore and a 3’ quencher.
  • The Tm of LNA® probes is fine-tuned by changing the number or position of LNAs® within the probe to achieve the desired specificity and stability.
• MGB (minor groove binding) probes generally produce high fluorescent background and are not recommended.
• Internal quenchers (i.e. ZEN™ or TAO™), are optional but may be included in the probe design to further quench background signal; they are beneficial for some assays.
• All probes and PAs should be ordered with HPLC purification.

Probe additive fundamentals

HPs (sometimes referred to as dual-labeled probes) carry a quencher at the 3’ end to suppress the fluorescence from the 5’ fluorophore when not incorporated by DNA polymerase. However, the quenching efficiency varies among probes, leading to inconsistent background fluorescence. On the Countable platform, background fluorescence from inefficient probe quenching reduces the ratio of signal-to-background fluorescence intensities and affects counting accuracy.

To mitigate this and ensure reliable data on the Countable platform, we strongly recommend adding a PA to your existing HP-based assay.

Design a probe additive

To design a PA, use the following guidelines:

1. Truncate the HP sequence from the 3’ end to generate a 10–14 bp fragment, ensuring the 5’ sequence is retained.
2. Estimate melting temperature (Tm) using an oligo analysis tool (e.g., IDT OligoAnalyzer™ Tool).
3. Select a truncated sequence with a Tm of 35–45 °C.
4. Generate the reverse complement of the truncated sequence.
5. Append the appropriate 3’ quencher matched to the 5’ fluorophore (see below). The resultant oligo is your PA.

Probe additive example

Order a probe additive