What is the recommended hybridization temperature relative to the probe Tm?

The recommended hybridization temperature is typically set to be 5°C below the melting temperature (Tm) of the probe. This is done to ensure that the conditions are favorable for the specific binding of the probe to its complementary target sequence.

Setting the hybridization temperature lower than the Tm helps to facilitate the formation of stable hybrid complexes, allowing for sensitive detection of the targeted sequence in the presence of potentially competing non-specific interactions. At temperatures equal to or above the Tm, the hybridization can become less specific, resulting in higher background noise and lower assay sensitivity.

In this context, hybridizing at 5°C below the Tm provides a balance between specificity and stability, allowing for effective results in molecular diagnostics.