DNA Melting Temperature Calculator

Find the melting temperature of a DNA primer with the Wallace rule or the GC formula, and see the GC content and annealing point.

What it does

The melting temperature of a DNA primer is the point at which half of the double helix falls apart into single strands. Every primer design starts with that number, because the annealing step of a PCR run has to sit just below it or the primer will not bind. The training material for DNA amplification from the National Institute of Justice puts the useful band at 52 to 58 degrees C for the melting temperature of a working primer, and warns that a primer with a melting temperature above 65 degrees C tends to stick to itself.

The oldest shortcut is the Wallace rule, and it is still the one most people quote. Every A and T pair adds 2 degrees C and every G and C pair adds 4 degrees C, because a G and C base pair stacks three hydrogen bonds where an A and T pair stacks two. The method needs nothing but the sequence, which is why it stays popular for quick checks and for teaching.

Longer primers need more heat to come apart, and GC rich primers need more heat than AT rich primers of the same length, so the formula was later rewritten to say that directly. The version from Marmur and Doty, published in 1962 in the Journal of Molecular Biology, reads the melting temperature off the percentage of GC bases and the length of the strand, and a salt corrected version adds the contribution of the ions in the buffer. Both are documented in the Biopython melting temperature module, which is the reference implementation many labs compare their own numbers against.

This page runs all three versions on the same sequence so the differences are visible rather than hidden. The Wallace rule gives the quick classroom answer. The GC formula gives a better estimate for a real primer, and the salt corrected version moves the number again when you tell it how much salt sits in the tube. Every primer also comes with a suggested annealing temperature five degrees below the melting point.

None of these formulas is exact. They are estimates that ignore the order of the bases, the stacking energy of neighbouring pairs, the concentration of the strands and the divalent ions in the mix. Programs such as Primer3 work from nearest neighbour thermodynamics instead and are what most laboratories design primers with. The value of this page is that it shows the classic numbers and the reasoning behind them, and it is fast enough to use while sketching a set of primers.

How to use

1. Pick the temperature scale you want to read the answer in.
2. Choose the formula: the Wallace rule for a quick check, the GC formula for a better estimate, or the salt corrected version when you know the buffer.
3. Paste or type the primer sequence using the letters A, C, G, T and U. Spaces are ignored and lowercase letters are accepted.
4. If you chose the salt corrected formula, enter the monovalent salt concentration in millimolar, usually 50 mM for a standard PCR buffer.
5. Read the melting temperature, then set the PCR annealing temperature about 5 degrees below it.
6. Check the GC content as well. A primer near 50 % GC is usually the easiest one to design, and a very long or very short primer will melt outside the useful band.

Formula

Tm

Melting temperature of the duplex in degrees C.

A, T, G, C

Counts of each base in the sequence.

%GC

Share of the bases that are G or C, from 0 to 100.

N

Length of the sequence in bases.

[Na+]

Monovalent salt concentration in moles per litre, so 50 mM is 0.05.

The salt term is the Schildkraut and Lifson correction, so it is the molar concentration that goes into the logarithm, not the millimolar one. All three formulas ignore base order, which matters: two primers with the same length and the same GC content can differ by several degrees in real melting point. A U in the sequence is counted as a T, since the two bases pair with the same strength.

Worked example

Reference table

Limits

• The formulas ignore the order of the bases, so two primers with the same length and GC content can melt several degrees apart in the tube.
• No nearest neighbour term is used, so hairpins, self complementary stretches and primer dimers are not detected.
• Only monovalent salt is handled. Magnesium and other divalent ions raise the melting point, and the salt corrected formula here does not model them.
• The numbers are estimates for primer design, not a substitute for running the primer on the real template.

Common mistakes

• Putting the salt concentration in millimolar into the logarithm. The correction needs the molar value, so 50 mM has to be written as 0.05.
• Using the Wallace rule on a long probe and expecting the same answer as the GC formula. The two rules were never meant to agree for long strands.
• Reading the annealing temperature as the melting temperature. Annealing runs cooler, usually 3 to 5 degrees below, or the primer will not pair with the template in time.
• Forgetting that every C and G adds 2 degrees C more than every A and T, so swapping a single base can move the melting point by 2 degrees.
• Choosing a primer with a very high GC content and expecting it to be specific. GC rich primers bind well but also stick to each other.

Try it yourself

Quick checks. Answers sit tucked under each question.

Questions

Sources

• National Institute of Justice: DNA amplification, primer melting temperature
• Biopython documentation: Bio.SeqUtils.MeltingTemp, the Wallace rule and the GC formulas
• Marmur J and Doty P, Journal of Molecular Biology 1962: base composition from thermal denaturation temperature

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