DNA Melting Temperature Calculator
Estimate the melting temperature (Tm) of a DNA primer from its sequence.
Only A, T, G and C bases are counted (case-insensitive). Any other characters are ignored.
Estimated Melting Temperature (Tm)
°C
Sequence Length (n)
GC Content
Wallace Rule Tm
GC% Formula Tm
Base Composition
What Is DNA Melting Temperature?
The melting temperature (Tm) of a DNA sequence is the temperature at which half of the double-stranded DNA molecules in a sample have separated (denatured) into single strands. It is a key property of any primer or oligonucleotide because the strength of base pairing determines how easily the two strands come apart when heated.
G–C base pairs are held together by three hydrogen bonds, while A–T pairs share only two. Sequences richer in G and C therefore tend to have a higher Tm. Sequence length also matters: longer duplexes are more stable and melt at higher temperatures.
Wallace Rule vs the GC% Formula
Wallace Rule (short oligos, n < 14)
Also called the "2+4 rule", it gives a quick estimate for short primers:
Tm = 2 × (A + T) + 4 × (G + C) °C
It is simple and works reasonably well for oligos shorter than about 14 bases, but it ignores salt concentration, oligo concentration and longer-range structure, so it becomes unreliable for longer sequences.
GC% Formula (longer sequences, n ≥ 14)
For longer sequences a GC-content based formula gives a better estimate:
Tm = 64.9 + 41 × (G + C − 16.4) / (A + T + G + C) °C
This formula accounts for both length and GC content. It is still an approximation: it does not model salt or strand concentration, and for precise work researchers use nearest-neighbour thermodynamic models.
Tm in PCR Primer Design
In a polymerase chain reaction (PCR), the annealing temperature is usually set a few degrees below the Tm of the primers. Choosing a good Tm helps primers bind specifically to their target.
- Aim for primers with a Tm in roughly the 52–62 °C range for standard PCR.
- Keep the Tm of a forward and reverse primer pair within about 5 °C of each other.
- Annealing temperature is often set about 3–5 °C below the lower primer Tm.
- Very high GC content can cause stable secondary structures; very low GC can reduce specificity.
Related Calculators
Educational Disclaimer: This DNA melting temperature calculator provides approximate estimates based on simplified formulas (the Wallace rule and a GC-content formula). These do not account for salt concentration, oligonucleotide concentration, mismatches or secondary structure. For experimental design requiring high accuracy, use nearest-neighbour thermodynamic models and validated laboratory protocols.