DNA Melting Temperature Calculator
The melting temperature of a DNA primer from its sequence — the Wallace rule for short oligos and the GC-content formula for longer ones — with the annealing temperature to use in a PCR.
DNA strands separate at a temperature set by their length and composition: G–C pairs have three hydrogen bonds, A–T pairs two, so GC-rich sequences melt hotter.
How the dna melting temperature calculator works
DNA strands separate at a temperature set by their length and composition: G–C pairs have three hydrogen bonds, A–T pairs two, so GC-rich sequences melt hotter. For primers under 14 bases the Wallace rule — 2 °C per A or T, 4 °C per G or C — is good enough; longer primers use the salt-adjusted GC formula.
The annealing temperature is a few degrees below the lower of the two primers' Tm. Too high and nothing binds; too low and everything does.
Formula: Tm (short) = 2(A+T) + 4(G+C); Tm (long) = 64.9 + 41 × (G+C − 16.4) / N
Worked examples
| Inputs | Melting temperature (°C) | Note |
|---|---|---|
| A 20-mer at 55% GC | 53.83 | about 54 °C |
| A short oligo | 30 | Wallace rule applies |
| AT-rich | 31.28 | a low Tm |
FAQFrequently asked questions
What is Tm?
The temperature at which half the DNA duplex has separated into single strands. It sets the annealing temperature for PCR and hybridization.
Why do GC-rich sequences melt hotter?
Three hydrogen bonds per G–C pair against two per A–T, plus stronger base stacking. Each GC adds about 4 °C in a short oligo.
Which formula should I trust?
For real primer design, nearest-neighbour thermodynamics with your actual salt and primer concentrations. These two are the quick rules that get within a few degrees.
What annealing temperature should I use?
Three to five degrees below the lower of the two primer Tms, then optimize with a gradient. Keep the two primers within about 5 °C of each other.
What length should a primer be?
Eighteen to thirty bases, 40 to 60% GC, and a G or C at the 3′ end. Shorter binds non-specifically; longer is slower and prone to hairpins.
Where these figures come from
- NCBI Bookshelf — Molecular Biology of the Cell — growth kinetics and molecular conventions
- National Human Genome Research Institute — the US genomics research institute
Last checked: September 2026. Formulas are the standard textbook forms; assumptions are stated on each page because they are where these models break.