Input
Result
Enter a primer sequence.
Method: SantaLucia 1998 unified nearest-neighbour ΔH°/ΔS°, helix initiation at each terminus, symmetry term for self-complementary strands, Ct/4 (or Ct) in the mass-action term, then the selected salt correction. Limit: a perfectly matched DNA/DNA duplex is assumed. Mismatches, dangling ends, hairpins and primer dimers are not modelled, and a Tm is not defined for ambiguous bases.
METHOD
How it is calculated
The duplex is treated as two states, single strands and a fully paired helix. Its enthalpy and entropy are sums of nearest-neighbour stacks plus helix initiation, taken from the SantaLucia (1998) unified parameter set, which is referenced to 1 M NaCl.
| Stack (5′→3′ / 3′→5′) | ΔH°, kcal/mol | ΔS°, cal/(mol·K) |
|---|---|---|
| AA/TT | -7.9 | -22.2 |
| AT/TA | -7.2 | -20.4 |
| TA/AT | -7.2 | -21.3 |
| CA/GT | -8.5 | -22.7 |
| GT/CA | -8.4 | -22.4 |
| CT/GA | -7.8 | -21.0 |
| GA/CT | -8.2 | -22.2 |
| CG/GC | -10.6 | -27.2 |
| GC/CG | -9.8 | -24.4 |
| GG/CC | -8.0 | -19.9 |
| Initiation, terminal G·C (each end) | 0.1 | −2.8 |
| Initiation, terminal A·T (each end) | 2.3 | 4.1 |
| Symmetry, self-complementary duplex only | 0 | −1.4 |
SantaLucia (1998), Table 1. Ct is the total strand concentration you enter as "Oligo, nM".
Salt corrections
f_GC is the GC fraction and N the primer length. The 3′-end ΔG°37 sums ΔG° = ΔH° − 310.15·ΔS° over the four stacks inside the last five bases (no initiation term), a measure of how stably the 3′ end pairs.
Which calculation this page matches
Every salt option reproduces Biopython's Bio.SeqUtils.MeltingTemp.Tm_NN with nn_table=DNA_NN3 and saltcorr 6, 5 or 7, with dnac1 = dnac2 = Ct/2 (or dnac1 = Ct and selfcomp=True for a palindrome). Calculators that use another parameter set (Breslauer 1986, SantaLucia & Hicks 2004), another concentration convention, or a different Mg2+ treatment will report different numbers for the same primer; compare like with like.
References
- SantaLucia J Jr (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 95:1460-1465. doi:10.1073/pnas.95.4.1460
- Owczarzy R, You Y, Moreira BG, Manthey JA, Huang L, Behlke MA, Walder JA (2004) Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures. Biochemistry 43:3537-3554. doi:10.1021/bi034621r
- Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA (2008) Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry 47:5336-5353. doi:10.1021/bi702363u
- von Ahsen N, Wittwer CT, Schütz E (2001) Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg2+, deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to empirical formulas. Clin Chem 47:1956-1961. doi:10.1093/clinchem/47.11.1956
WORKED EXAMPLE
A worked example, computed by this page
Primer 1 GTAAAACGACGGCCAGT (17 nt, GC 52.9 %) and primer 2 CAGGAAACAGCTATGAC, at 250 nM oligo, 50 mM Na+, 1.5 mM Mg2+ and 0.8 mM total dNTP. These are the values the calculator above starts with.
| Salt correction | Primer 1 Tm, °C | Primer 2 Tm, °C | ΔTm, °C |
|---|---|---|---|
| Owczarzy 2004 | 58.8 | 52.9 | 5.9 |
| SantaLucia 1998 | 56.9 | 51.3 | 5.6 |
| Owczarzy 2008 | 55.9 | 50.2 | 5.7 |
The spread between rows is the choice of salt model alone, on identical stacks. A palindrome behaves differently: GAATTC is its own complement, takes the symmetry term and uses Ct rather than Ct/4, and under the same conditions gives -11.1 °C.
WHERE THIS RUNS
The calculation runs in your browser
The arithmetic on this page is JavaScript executing in your browser. The sequence you enter is not sent anywhere: the calculator makes no network request with it, and there is no account, upload or server-side step. You can disconnect from the network after the page has loaded and the calculator keeps working.