DNA Melting Temperature (Tm) Calculator
Enter a short DNA sequence to compute its melting temperature (Tm) using the Wallace rule, the classic formula for estimating when a short double-stranded DNA sequence separates into single strands. Two 3D diagrams compare a low-Tm and high-Tm double helix, and charts show how Tm scales with GC content and sequence length.
Reviewed by the ToolNestr Editorial Team — July 2026
Low-Tm vs high-Tm double helix
1. An AT-rich, low-Tm helix (loosely bonded)
Only two hydrogen bonds per base pair — this helix separates at a relatively low temperature.
2. A GC-rich, high-Tm helix (tightly bonded)
Three hydrogen bonds per base pair — this helix requires more heat to separate into single strands.
Melting temperature graphs
How it works
The core idea in one line: a G-C base pair is held together by three hydrogen bonds while an A-T pair has only two, so every G or C in a short DNA sequence contributes more thermal stability than every A or T — a fact the Wallace rule turns directly into a simple per-base temperature contribution.
Tm = 4°C × (G+C) + 2°C × (A+T)
Wallace rule — valid for short oligonucleotides (<14 bases)
The Wallace rule assigns a fixed temperature contribution to each base based purely on its hydrogen-bonding strength: 4°C for every G or C (three hydrogen bonds each) and 2°C for every A or T (two hydrogen bonds each), then sums these contributions across the whole short sequence. This simple additive model works well for short oligonucleotides where neighboring-base interactions have limited room to matter, which is exactly the length range (roughly under 14 bases) most relevant to PCR primer design.
Worked example 1 — a GC-balanced 10-base primer
Given: DNA sequence: GCGCATGCAT (10 bases)
This gives a quick estimate suitable for designing a PCR primer annealing step, though real-world protocols also apply a rule-of-thumb offset below Tm for the actual annealing temperature.
Worked example 2 — a GC-rich short sequence
Given: DNA sequence: GCGCGCCGGC (10 bases)
Compared to the balanced example above, this fully GC sequence has a notably higher Tm (40°C vs 32°C) — a direct consequence of G-C pairs' extra hydrogen bond.
How GC content affects Tm for a 10-base sequence
Every base pair matters — replacing an A-T pair with a G-C pair raises the Wallace-rule Tm estimate by exactly 2°C per swap.
| GC count (of 10) | AT count | Tm |
|---|---|---|
| 0 | 10 | 20°C |
| 4 | 6 | 28°C |
| 6 ★ | 4 | 32°C |
| 10 | 0 | 40°C |
★ Reference row (worked example 1). The Wallace rule's simplicity — a fixed +4°C per G/C base and +2°C per A/T base — makes it fast for rough estimates, though it ignores base order and neighboring-base effects.
Where DNA melting temperature actually matters
🧪 PCR primer design
Molecular biologists calculate primer Tm to choose an appropriate annealing temperature for PCR, typically setting the actual annealing temperature a few degrees below the calculated Tm.
🔬 DNA probe and hybridization assay design
Techniques like Southern blotting and fluorescent in situ hybridization (FISH) depend on carefully matched probe Tm values to ensure specific, reliable binding to target sequences.
🧬 Restriction enzyme reaction optimization
Understanding local DNA melting behavior helps researchers optimize conditions for enzymes that require specific double-strand or single-strand DNA states to function.
🎓 Teaching DNA thermodynamics
The Wallace rule is often students' first quantitative introduction to how base composition determines a physical property of DNA, bridging chemistry and molecular biology concepts.
Common misconceptions
"The Wallace rule accurately predicts Tm for any length of DNA sequence."
The Wallace rule is only accurate for very short oligonucleotides (under about 14 bases) — for longer sequences, more sophisticated formulas incorporating nearest-neighbor thermodynamics and salt concentration are needed for accurate predictions.
"Two sequences with the same GC content always have the same Tm."
The Wallace rule treats all G/C and A/T bases as contributing equally regardless of order, but more precise nearest-neighbor models show that the actual arrangement of bases (not just the overall GC%) does affect real melting behavior, especially for longer sequences.
"Melting temperature means the DNA physically melts like ice, into a liquid."
"Melting" in this context refers specifically to the double helix separating into two single strands (denaturation) — the DNA doesn't change physical state like ice to water; the two strands simply come apart from each other.
"Higher Tm always makes a PCR reaction work better."
PCR primers need a Tm in a sensible, moderate range (commonly 50-65°C) and — just as importantly — the two primers in a reaction should have SIMILAR Tm values to each other, not simply the highest possible Tm.
Formula sources & further reading
The formulas here are standard, traceable to:
- • OpenStax, Biology 2e — Chapter 14, "DNA Structure and Function" (free, peer-reviewed). openstax.org
- • Wallace, Shaffer, et al. (1979) — "Hybridization of synthetic oligodeoxyribonucleotides," Nucleic Acids Research (original Wallace rule reference).
- • Alberts et al., Molecular Biology of the Cell — Chapter 4, DNA, Chromosomes, and Genomes.
Tm = 4(G+C) + 2(A+T), the Wallace rule, valid for short oligonucleotides under ~14 bases. Longer sequences need nearest-neighbor thermodynamic models for accuracy.
How to use this calculator
Enter a short DNA sequence
Type a sequence under about 14 bases for the Wallace rule to apply accurately.
Read the melting temperature
Tm solves instantly based on the base composition.
Check the base breakdown
See exactly how many G/C versus A/T bases are driving the result.
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Frequently asked questions
What is DNA melting temperature (Tm)?
Melting temperature (Tm) is the temperature at which half of a double-stranded DNA sequence has separated into single strands. It depends primarily on sequence length and GC content.
What is the Wallace rule?
The Wallace rule is a simple formula for estimating Tm in short oligonucleotides (under about 14 bases): Tm = 4°C × (G+C count) + 2°C × (A+T count).
Why does GC content raise melting temperature?
G-C base pairs form three hydrogen bonds, while A-T pairs form only two — more hydrogen bonds means more energy (heat) is needed to break them apart, so GC-rich sequences have a higher melting temperature.
Does the Wallace rule work for long DNA sequences?
No — the Wallace rule is only accurate for short oligonucleotides (typically under 14 bases), like PCR primers. Longer sequences require more sophisticated formulas that account for nearest-neighbor base-stacking interactions and salt concentration.
How is Tm used in molecular biology techniques?
Tm is central to PCR primer design (setting the annealing temperature), DNA hybridization assays, and any technique requiring controlled separation and reannealing of DNA strands.