DNA melting temperature (Tm) calculator

Paste a primer or probe. Two estimates appear: the nearest-neighbour Tm, which accounts for the order of the bases, the salt and the strand concentration, and the basic formula that most quick calculators use.

Nearest-neighbour method (SantaLucia 1998)

ΔH° = Σ ΔH°(pairs) + ΔH°(initiation) ΔS°(Na⁺) = ΔS°(1 M NaCl) + 0.368 × (N − 1) × ln[Na⁺]

Tm = 1000 ΔH° ÷ (ΔS° + R ln(CT ÷ 4)) − 273.15 (°C, ΔH° in kcal/mol, ΔS° in cal/(K·mol), R = 1.9872)

Each of the ten distinct Watson–Crick nearest-neighbour stacks has a measured enthalpy and entropy (the unified parameters, 1 M NaCl), plus initiation terms for a terminal G·C or A·T pair. Sodium stabilises the duplex through the entropy correction. CT is the total strand concentration: with equal amounts of two strands at c each, CT = 2c and CT/4 = c/2. A self-complementary sequence forms the duplex with itself, so CT/4 becomes CT and a symmetry term is added.

Basic formulas

Under 14 bases: Tm = 2 (A + T) + 4 (G + C) (Wallace et al. 1979)

14 bases or more: Tm = 64.9 + 41 (G + C − 16.4) ÷ N

Limits

• Assumes a perfectly matched duplex that melts in two states, sodium as the only cation and no denaturants. Mg²⁺ in PCR buffers raises Tm and dNTPs bind it back; neither is modelled here, so PCR annealing temperatures are usually set from a polymerase maker’s calculator or a few degrees below Tm.
• Mismatches, dangling ends, modified bases and secondary structure (hairpins, primer dimers) are not considered.
• The basic formulas ignore salt and sequence order and can be several degrees off.

Worked example

5′-CGTTGA-3′ in 1 M NaCl: ΔH° = −10.6 − 8.4 − 7.9 − 8.5 − 8.2 + 0.1 + 2.3 = −41.2 kcal/mol and ΔS° = −115.4 cal/(K·mol), so ΔG°37 = −5.41 kcal/mol (SantaLucia’s worked example gives −5.35 from the rounded ΔG° table). For the 24-mer shown, 50 mM Na⁺ and 250 nM of each strand give a nearest-neighbour Tm of 57.5 °C.

References

• SantaLucia, A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics, PNAS 95, 1460–1465 (1998)
• Wallace et al., Hybridization of synthetic oligodeoxyribonucleotides to ΦX174 DNA: the effect of single base pair mismatch, Nucleic Acids Res. 6, 3543–3557 (1979)
• Kibbe, OligoCalc: an online oligonucleotide properties calculator, Nucleic Acids Res. 35, W43–W46 (2007)