Oligo Analyzer – Tm & Dimer Calculator
Nearest-neighbor Tm, ΔG, hairpins and primer dimers for any oligo or primer pair.
A nearest-neighbor oligo analyzer for PCR primers: paste a single oligo, a forward/reverse pair, or a whole list, and get SantaLucia 1998 Tm, GC%, and dH/dS/dG37, plus a primer dimer and hairpin screen covering self-dimers and cross-dimers, each drawn as a base-paired alignment. Set the oligo, Na+, Mg2+ and dNTP concentrations to match your reaction; batch results export as CSV.
Paste one oligo, a primer pair or a list of oligos.
Paste an oligo above for its Tm, GC content, hairpin and self-dimer. Two oligos are also checked against each other; three or more are reported as a table.
Tm uses SantaLucia (1998) nearest-neighbor thermodynamics, with a salt correction for Na⁺ and a monovalent equivalent for Mg²⁺ and dNTP. Hairpin and dimer ΔG are screening estimates; confirm critical designs with a dedicated secondary-structure tool. A dimer or hairpin at the 3′ end is the most likely to disrupt PCR.
A whole set of oligos, every pair
Screens every pair in a multiplex panel or primer list for cross-dimers, up to 384 oligos. Duplicate sequences are flagged by name, the Tm spread across the set is reported, and a 3′-end pairing is held to a stricter threshold than one that only sequesters a primer internally.
▸Conflict thresholds: -6 kcal/mol at a 3′ end · -9 internal
A pairing at an oligo's 3′ end, where extension starts, counts as a conflict at a weaker ΔG than one that only sequesters the primer internally. Defaults are −6 and −9 kcal/mol.
Names are taken from FASTA headers or “name,sequence” rows; bare sequences, one per line, are named oligo_1, oligo_2, ….
Paste two or more oligos in the box above to screen every pair in the set.
The full folding model, for one oligo or two
Folds one oligo, or two together, with ViennaRNA’s published loop model at the chosen temperature and salt, and reports the minimum-free-energy structure: loops, bulges and dot-bracket notation. The nearest-neighbor screen above reports only the most stable pairing.
Paste an oligo in the box at the top of this page and it is folded here.
One oligo or two, at most 200 nt each — the loop model is for oligos, not templates, and a longer strand is refused.
A whole multiplex panel, not just its oligos
Checks a multiplex PCR panel as a whole: the Tm spread across targets, which must allow a single annealing temperature; whether the product sizes resolve on a gel; and which primer pairs cannot share a tube.
Design, edit, and verify complete constructsSeqStudio combines sequence editing and annotation, plasmid maps, primer/cloning/CRISPR design, Sanger verification, and GenBank/SnapGene files in one workspace.
How to use the Oligo Analyzer tool
- 1The Tm is computed at the conditions named on the Conditions line under the box: 250 nM oligo, 50 mM Na+, 1.5 mM Mg2+, 0.2 mM dNTP by default. Open that line to change them by hand or pick the qPCR or Isothermal / Gibson preset.
- 2Paste what you have into the one box: a single oligo, a forward/reverse pair, or a whole list as one per line, FASTA, or "name,sequence" rows. There is no mode to pick; the page counts what you pasted and reports what it read.
- 3Read the results: one oligo gives Tm (NN), dG37 and Hairpin / Self-dimer cards; two are checked as a PAIR, adding each Tm, the delta-Tm (flagged above 5 C) and the cross-dimer between them, with the per-oligo detail one click down; three or more come back as a table with an Export CSV button. A dG below about -6 kcal/mol, or any structure marked "involves 3' end", is the one to examine first.
Frequently asked questions
How is the nearest-neighbor Tm calculated?
Using SantaLucia (1998) unified nearest-neighbor ΔH/ΔS parameters, with an entropy salt correction for Na⁺ and a monovalent-equivalent for Mg²⁺ (corrected for dNTP chelation). It is more accurate than the Wallace or salt-adjusted formulas, especially for primer-length oligos.
What counts as a problematic dimer or hairpin?
A more negative ΔG means a more stable structure. As a rough guide, ΔG below about −6 kcal/mol merits attention, and any structure that involves the 3′ end is the most likely to interfere with extension. These are screening estimates; confirm critical designs with a dedicated folding tool.
Can I analyze many oligos at once?
Yes. Batch mode accepts one oligo per line, FASTA, or “name,sequence” rows, computes Tm, GC, ΔG and structure for each, and exports a CSV.