DNA Melting Temperature Calculator
The melting temperature (Tm) of a DNA oligonucleotide is the temperature at which 50% of the double-stranded DNA is denatured into single strands. Tm is critical for designing PCR primers, hybridization probes, and restriction enzyme analyses. This calculator uses the Wallace rule (Tm = 2(A+T) + 4(G+C)) for oligonucleotides of 14 to 20 nucleotides. Enter your sequence in the 5-to-3 direction using the standard IUPAC bases A, T, G, and C. The calculator counts each nucleotide and applies the formula from the original Wallace et al. 1979 publication, one of the most widely cited oligonucleotide Tm references.
Wallace rule formula
Tm = 2(A + T) + 4(G + C) °C
Where A, T, G, and C are the counts of adenine, thymine, guanine, and cytosine residues in the oligonucleotide. This rule is valid for short oligonucleotides (14 to 20 bases) in 1 M NaCl. Source: Wallace et al. (1979), Nucleic Acids Research.
Nearest-neighbor correction (salt-adjusted)
For longer sequences or when working at non-standard salt concentrations, a salt-correction is applied to the basic Tm. A common approximation is:
Tm(corrected) = Tm(Wallace) - 16.6 * log10(0.05 / [Na+])
Where [Na+] is the molar sodium concentration. At 50 mM NaCl the correction is approximately -16.6 * log10(0.05/0.05) = 0 degrees; at 1 mM NaCl the Tm drops by about 33 degrees compared to 1 M NaCl conditions.
GC content and Tm
- GC pairs form 3 hydrogen bonds; AT pairs form 2. Higher GC content raises Tm.
- For primers, aim for 50 to 60% GC content and a Tm of 55 to 65 degrees Celsius.
- Keep both forward and reverse primer Tm values within 5 degrees of each other.
- Avoid runs of more than 4 identical bases or self-complementary 3-prime ends.
DNA melting temperature: frequently asked questions
What is the DNA melting temperature (Tm)?
The melting temperature (Tm) is the temperature at which 50% of a DNA duplex is in single-stranded form. It depends on sequence length, GC content, salt concentration, and oligonucleotide concentration.
What is the Wallace rule for calculating Tm?
The Wallace rule estimates Tm as: Tm = 2(A+T) + 4(G+C) degrees Celsius, where A, T, G, and C are the counts of each nucleotide. This approximation is reliable for oligonucleotides of 14 to 20 bases.
When should I use the nearest-neighbor method instead of the Wallace rule?
The nearest-neighbor method is more accurate for oligonucleotides shorter than 14 bases or longer than 20 bases, and when you need to account for salt concentration and strand concentration effects.
Why does GC content affect melting temperature?
GC base pairs form three hydrogen bonds while AT base pairs form only two. More GC pairs therefore require more energy (higher temperature) to separate the strands, raising the Tm.
What is a typical PCR primer Tm range?
For most PCR applications, primer Tm values between 55 and 65 degrees Celsius are recommended, with both forward and reverse primers within 5 degrees of each other to ensure balanced amplification.
Official sources
- Wallace R.B. et al. (1979). Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: Nucleic Acids Research, PubMed Central.
- NCBI Primer-BLAST documentation: NCBI Primer Design.
Reviewed by the CalculatorHub team, edited by James Graham, 14 June 2026. See our methodology.