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Bioinformatics & Sequence Analysis

DNA Nearest-Neighbour Melting-Temperature Workbench

Estimate perfect-match DNA/DNA duplex melting with the SantaLucia unified nearest-neighbour parameters, explicit strand concentration, sodium-ion concentration, terminal terms and symmetry correction.

Biology · experimental measurements

Expose every thermodynamic contribution and model boundary instead of returning a primer temperature detached from concentration, sodium and sequence assumptions.

Private calculations in your browser · explicit inputs and model boundaries
Example preview · Primer-like DNAMagnitude of stack enthalpy contributions
1–2 · AT7.2 kcal/mol
2–3 · TG8.5 kcal/mol
3–4 · GC9.8 kcal/mol
4–5 · CG10.6 kcal/mol
5–6 · GT8.4 kcal/mol
6–7 · TA7.2 kcal/mol
7–8 · AC8.4 kcal/mol
8–9 · CG10.6 kcal/mol
9–10 · GT8.4 kcal/mol
10–11 · TT7.9 kcal/mol
11–12 · TA7.2 kcal/mol
12–13 · AG7.8 kcal/mol
13–14 · GC9.8 kcal/mol

Bars show the magnitude of every adjacent-stack ΔH contribution. The full signed terms plus initiation, ends, symmetry and sodium correction remain in the ledger.

  1. 1EnterProvide the known values
  2. 2CalculateResults update automatically
  3. 3VerifyReview the details and units
Try an example

Exact A/C/G/T only; the model assumes a perfectly complementary DNA strand.

Enter Na+ explicitly. Potassium, magnesium and mixed-ion solutions are outside this correction.

Calculation result

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Understand the relationship

The reasoning behind the result

Nearest neighbours carry sequence context

ΔH = ΔHinit + ΣΔHstack + terminal terms

Each adjacent base-pair stack contributes empirical enthalpy and entropy. The unified model uses ten independent Watson–Crick nearest-neighbour classes plus initiation and terminal terms.

Reordering a sequence can change the estimate even when total GC content is unchanged.

Concentration changes bimolecular melting

non-self denominator uses ln(Ct/4)

A non-self-complementary duplex formed from equal strand concentrations uses the Ct/4 convention. A self-complementary sequence uses Ct and receives the entropy symmetry correction.

The workbench detects exact reverse-complement identity and displays the applied convention.

Sodium correction is model specific

ΔSNa = ΔS1M + 0.368(N−1)ln[Na+]

This empirical length-dependent correction is stated for entered sodium-ion concentration and belongs to the selected unified model.

It does not treat potassium, magnesium, dNTPs or mixed-ion chemistry as interchangeable with sodium.

A calculated Tm is conditional

Mismatches, dangling ends, chemical modifications, secondary structure, crowding and instrument protocol can change measured melting behavior.

The estimate is not a PCR annealing-temperature prescription and does not certify primer specificity or assay performance.

Follow the numbers

Calculate a primer-like DNA duplex estimate

  1. Read every adjacent two-base step in ATGCGTACGTTAGC.
  2. Sum the unified ΔH and ΔS stack terms with initiation and terminal A/T contributions.
  3. Detect that the oligonucleotide is not self-complementary, so the concentration term uses Ct/4.
  4. Apply the length-dependent entropy correction at 0.05 M entered sodium ions.
  5. Evaluate the displayed thermodynamic denominator and convert kelvin to degrees Celsius.

The result is conditional on a perfect-match DNA/DNA two-state model and the entered solution quantities.

Quick guide

How to use this calculator

  1. Enter the exact DNA oligonucleotide and the total strand concentration used by this convention.
  2. Enter a positive sodium-ion concentration in the selected unit.
  3. Inspect every nearest-neighbour, initiation, terminal, symmetry and sodium term before comparing the estimate with another tool or experiment.

Calculation method

Calculation and interpretation

Expose every thermodynamic contribution and model boundary instead of returning a primer temperature detached from concentration, sodium and sequence assumptions.

Tm = 1000ΔH/[ΔS([Na+]) + R ln(Ct/f)] − 273.15, with f=1 for self-complementary and f=4 for non-self-complementary equal-concentration strands; ΔS sodium correction = 0.368(N−1)ln[Na+].

Worked example

Calculate a primer-like DNA duplex estimate

The result is conditional on a perfect-match DNA/DNA two-state model and the entered solution quantities.

Tm = 1000ΔH/[ΔS([Na+]) + R ln(Ct/f)] − 273.15, with f=1 for self-complementary and f=4 for non-self-complementary equal-concentration strands; ΔS sodium correction = 0.368(N−1)ln[Na+].

Supported inputs

Precision and limits

Perfect-match DNA/DNA only

The model accepts exact A/C/G/T and assumes its perfectly complementary unmodified DNA strand.

Unified parameter set

SantaLucia unified nearest-neighbour, initiation, terminal and symmetry terms are used as one named model.

Reported salt-fit envelope

The cited 1998 sodium correction was fit to 26 duplexes shorter than 17 base pairs at 0.01–0.3 M NaCl. The result explicitly marks longer sequences or sodium values outside that reported envelope as extrapolations.

Sodium-only correction

Potassium, magnesium, dNTPs, mixed ions and activity corrections are not inferred or converted.

No structure or mismatch model

Hairpins, dimers, dangling ends, mismatches and chemical modifications require additional parameters.

No assay prescription

The result does not select a PCR annealing temperature or establish specificity, yield or experimental melting behavior.

Computational acceptance

Exact DNA sequences of 2–60 bases, strand concentrations from 0.001 nM to 1 mM and sodium-ion concentrations from 0.1 mM to 2 M are algebraically accepted; this range is wider than the reported salt-fit envelope.

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