Melt temperature

Melt temperature (also Tm) is the temperature at which 50% of a double-stranded nucleic acid (usually DNA, but also RNA or any hybrid combination) is denatured. Melt temperature varies with base composition. For instance, a greater GC content typically increases Tm because more hydrogen bonds need disrupting in GC base-pairs (3 compared to 2 in AT pairs). Tm is also influenced by salt concentration (higher salt concentration stabilises hybrids, even when mismatched) and by formamide concentration (higher formamide concentration reduces Tm by disrupting hydrogen bonds)

In nucleic acid hybridisation, the Tm of the hybrid is an important factor for consideration. The Tm of a hybrid depends on its composition: DNA-DNA hybrids have the lowest Tm, while RNA-RNA hybrids have the highest (DNA-RNA hybrids are intermediate).

Tm can be calculated using the following formula:

Tm (degrees C) = 81.5 + 16.6 (log10Na+) + 0.4(%GC) - 0.72(% formamide) - 600/length

[latter term used only for fragments greater than 60 nts in length]

When calculating hybrid Tm for a shorter probe, such as a PCR primer, a simpler formula can be used, like so:

Tm (degrees C) = (2 x A + T) + (4 x G + C)

In PCR reactions, it is essential that both primers have the same Tm to ensure coordinated thermal cycling.