Melting Temperature (Tm)
The melting temperature (Tm) of a DNA duplex is the temperature at which half of the molecules are double-stranded and half have separated into single strands. It decides how hot a PCR must run for primers to bind (anneal) and how stable a hybridisation is.
Three ways to estimate it
The Wallace rule: Tm = 2 × (A + T) + 4 × (G + C), in °C. Quick enough for a mental estimate, but only reasonable for oligos shorter than about 14 bases.
The long-DNA formula: Tm = 81.5 + 16.6 × log10[Na⁺] + 0.41 × %GC − 675 ÷ N, where N is the length. It uses only composition and length, which suits long DNA (14 bases and up) but ignores base order.
Nearest-neighbor: the standard for primers. Stacking energy depends on each pair of adjacent bases, so the method adds up the enthalpy (ΔH) and entropy (ΔS) of every dinucleotide step, plus end corrections, and finds the temperature where half the strands are melted. The 1998 SantaLucia parameter set is the one used by most modern tools. It is valid for oligos of about 8 to 60 bases.
Two worked examples
Wallace rule. For the 10-base oligo ATGCGCATGC there are 4 A or T and 6 G or C. Tm = 2 × 4 + 4 × 6 = 8 + 24 = 32 °C.
Long-DNA formula. For 100 bases at 50% GC in 0.05 M Na⁺: Tm = 81.5 + 16.6 × log10(0.05) + 0.41 × 50 − 675 ÷ 100 = 81.5 − 21.6 + 20.5 − 6.75 ≈ 73.7 °C.
Why calculators disagree
Salt: more sodium, magnesium or other cations shield the phosphate backbone and raise Tm. Concentration: the more strand there is, the easier it is to find a partner and the higher the Tm. Parameter sets and salt corrections also differ between tools. Two calculators can differ by several degrees for the same primer, so compare numbers from the same method and settings.
From Tm to annealing temperature
A common starting point is an annealing temperature about 3–5 °C below the lower Tm of the two primers, then optimising with a temperature gradient. The two primers should have Tm values within 2–3 °C of each other so both bind well at one temperature. Mismatches, primer-dimers and hairpins reduce the effective Tm, which is why primer design checks for them.
How Aaron's Archive does it
| Item | What the tool does |
|---|---|
| Recommended value | Nearest-neighbor (SantaLucia 1998) for 8–60 base A/C/G/T oligos; the long-DNA formula for longer sequences; the Wallace rule is shown for reference. |
| Conditions | 50 mM monovalent salt and 250 nM oligo, the usual PCR-primer assumptions (1 M NaCl would read about 15 °C higher). Magnesium is not modelled. |
| Accuracy check | Across 500 random oligos the nearest-neighbor Tm agrees with the Primer3 library to within 0.053 °C at identical conditions. |
| RNA | RNA-specific parameters: nearest-neighbor values for short RNA and a long-RNA formula, since RNA duplexes are more stable than DNA ones. |
| Melting curve | A smooth two-state model drawn from the same thermodynamics, exactly 50% melted at the Tm. It is a model, not a measured melt. |
Pitfalls
The Tm of a whole gene is rarely what matters; for PCR it is the Tm of each primer. The tool reports the Tm of a perfectly matched duplex under simple conditions. Real reactions contain Mg²⁺, dNTPs, additives and mismatches that all shift it.
Common questions about melting temperature
What is Tm in PCR?
The temperature at which half of a primer is bound to its target and half is free. It guides the annealing temperature of the reaction.
How do I calculate the annealing temperature from Tm?
Start about 3 to 5 °C below the lower of the two primer Tm values, then adjust with a temperature gradient.
Why do two Tm calculators give different answers?
They use different methods, parameter sets and salt corrections. Compare numbers from the same method and settings.
Where can I calculate the Tm of my primers?
The PCR primer design tool reports the Tm of each primer it proposes, using nearest-neighbor values.