Biology · DNA
GC content and melting temperature.
Paste a DNA sequence to get its GC percentage, base counts and two quick melting temperature (Tm) estimates. Nothing you enter leaves this page.
GC content and the two Tm estimates
GC content is the percentage of bases in a DNA or RNA sequence that are guanine or cytosine. Each G-C pair is held by three hydrogen bonds and each A-T pair by two (Wikipedia, GC-content), which is why higher GC content generally means a higher melting temperature and why primers' GC content is used to predict annealing temperature. Bases other than A, C, G and T (or U, read as T) are ignored in the counts.
The Wallace rule is Tm = 4 °C × (G + C) + 2 °C × (A + T), a rule of thumb for primers of 14 to 20 nt (Biopython documentation). The salt-adjusted value uses the empirical formula Tm = 81.5 + 0.41 × (%GC) − 675 / N + 16.6 × log10[Na+], with N the length and [Na+] in mol/L (Marmur and Doty 1962; Schildkraut and Lifson 1965, as listed by Biopython).
Worked example. ACGTTGCAATGCCGTA is 16 nt with 8 G or C, so GC content is 50.0%. Wallace: 4 × 8 + 2 × 8 = 48 °C, the value Biopython's documentation gives for this exact sequence. Salt-adjusted at 50 mmol/L Na+: 81.5 + 20.5 − 42.19 + (−21.60) = 38.2 °C. The two estimates differ by about 10 °C, a reminder that both are rough; Biopython also offers a nearest-neighbour method for more careful work.
Frequently asked questions
How is GC content calculated?
GC content = (G + C) divided by the total number of A, C, G and T bases, times 100. This tool ignores characters that are not A, C, G, T or U.
What is the Wallace rule?
Tm = 4 C x (G + C) + 2 C x (A + T). Biopython describes it as a rule of thumb for primers of 14 to 20 nucleotides.
Why do the two Tm values differ?
They are different empirical shortcuts. The salt-adjusted formula accounts for length and sodium concentration, the Wallace rule does not, so they can disagree by several degrees.
Can I use these for ordering primers?
Treat them as first estimates only. Primer design usually uses nearest-neighbour thermodynamics and the exact buffer, so check with the supplier or a design tool before relying on a value.
Is my sequence uploaded anywhere?
No. The calculation runs in your browser, and this page loads no advertising or third-party scripts.
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Teaching model, not advice
Quick estimates for study and planning, not validated for primer, probe or assay design.