Drybench Sequence.
Primer check: Tm, dimers, hairpins and binding sites
Two primers checked for Tm and ΔTm, hairpins, self-dimers and the cross-dimer by SantaLucia and Hicks 2004, and binding sites on a template.
Sequence
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Answer
Primer check
Check two primers, inspect their binding sites and create the predicted PCR product.
More about this
Paste the forward primer’s annealing region in Sequence; enter both primers 5′ to 3′.
Plain DNA or one FASTA record, up to 50,000 bases. Supply the template separately from the forward primer.
Predicted PCR product
Exact matches of the two supplied annealing regions on this template only. The product is linear DNA, read 5′ to 3′ from the forward primer.
Add 5′ tails
Enter only the added 5′ bases here. Keep the matching annealing regions in the two primer fields. Tm uses the annealing regions; hairpin and dimer checks include the tails.
Add the template sequence to predict a product.
Sequence and provenance
FASTA contains the product sequence. GenBank also includes primer annotations and source details. The provenance file keeps the exact inputs and selected sites for review.
This predicts a sequence, not amplification or genomic specificity. Mismatched and overlapping sites, single-primer products and polymerase-added bases are not modelled.
Paste a sequence, or open an example, and every operation answers.
Open in the workbenchThe workbench carries this paste and offers every operation on it.
Worked example
- T7 promoter and T7 terminator primers at 250 nM in 50 mM Na⁺: Tm 47.6 and 54.5 °C, ΔTm 6.9 °C, annealing about 42.6 °C by the rule of thumb (5 °C below the lower Tm).
- Monovalent correction: Owczarzy 2004, fitted at 69 mM to 1.02 M Na⁺. It is also used from 20 to 69 mM, an extrapolation whose error has not been established here: Owczarzy 2008 places typical PCR buffers (20 to 100 mM monovalent) where this equation applies.
- The forward primer forms no hairpin with ΔG°37 below 0 at these conditions.
- The cross-dimer's most stable structure has ΔG°37 -2.89 kcal/mol (SantaLucia and Hicks 2004 rules; the special loop bonuses are not included).
- On a 372-bp template holding both sites the forward primer binds at 1 to 20 and the reverse on the other strand, giving a 372-bp product.
Method
ΔTm = |Tm₁ − Tm₂|; Ta = lower Tm − 5 °C (rule of thumb); hairpin and dimer ΔG°37 by the SantaLucia and Hicks 2004 rules, lowest ΔG°37 over every structure
- Oligo melting temperature by nearest neighbour. SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 1998;95:1460-1465 (Table 2; read 6 October 2026); concentration models after SantaLucia J Jr, Hicks D. Annu Rev Biophys Biomol Struct 2004;33:415-440.Checked against Biopython 1.88 MeltingTemp.Tm_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).
- Pair ΔTm and the annealing temperature rule of thumb. Arithmetic: ΔTm is the difference of the two Tm values; Ta about 5 °C below the lower Tm is a rule of thumb, not a prediction.
- Hairpin and dimer ΔG°37 by nearest-neighbour thermodynamics. SantaLucia J Jr, Hicks D. The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 2004;33:415-440; stacking from SantaLucia J Jr. Proc Natl Acad Sci USA 1998;95:1460-1465; dangling ends from Bommarito S, Peyret N, SantaLucia J Jr. Nucleic Acids Res 2000;28:1929-1934; salt as a sodium equivalent after von Ahsen N, Wittwer CT, Schütz E. Clin Chem 2001;47:1956-1961.Hairpins = ntthal 2.6.1 within 0.4 kcal/mol on ntthal's own structures, 15 fixtures (6 October 2026); dimers = ntthal 2.6.1 within 0.1 kcal/mol on ntthal's own structures, 16 fixtures (6 October 2026). ntthal reports the structure with the highest melting temperature and this card the lowest ΔG°37.
- Monovalent salt correction. Owczarzy R, You Y, Moreira BG, et al. Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures. Biochemistry 2004;43:3537-3554 (equation 22).Checked against Biopython 1.88 MeltingTemp.Tm_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).
- Magnesium and mixed-cation salt correction. Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry 2008;47:5336-5353 (equations 16 and 18 to 20).Checked against Biopython 1.88 MeltingTemp.Tm_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).
Constants
- santalucia-1998: SantaLucia 1998 unified nearest-neighbour ΔH° and ΔS°
- santalucia-hicks-2004: SantaLucia and Hicks 2004 loop, mismatch and dangling-end increments with the 1998 stacks
- owczarzy-2004: Owczarzy et al. 2004 monovalent correction, equation 22
- owczarzy-2008: Owczarzy et al. 2008 Mg²⁺ and mixed-cation correction, equations 16 and 18 to 20
Limits
- For research and education only, and not medical advice: nothing here is an assessment of any person's variant, sample or molecule, and a difference reported from a reference is a difference, not an interpretation.
- Tm and structure calculations require finite, nonnegative ionic concentrations and a positive model-supported ionic strength. Zero ionic strength is refused rather than silently treated as a salt correction of zero.
- pI, Tm and molecular weight depend on the method; the method and its parameter set are named on every card, and no alternative pK set is offered in this version.
- Tags and protease sites are found by their published motifs and a cut is proposed at the site nearest an end; confirm it, since a motif inside the protein is not a cleavage site.