Oligo Analyzer – Tm & Dimer Calculator
Nearest-neighbor Tm, ΔG, hairpins and primer dimers for any oligo or primer pair.
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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.
One oligo, a primer pair, or a whole list — it reads whichever you paste.
Paste an oligo above for its Tm, GC, hairpin and self-dimer. Two get checked against each other as well; three or more come back as a table.
Tm uses SantaLucia (1998) nearest-neighbor thermodynamics with a salt correction for Na⁺ and a monovalent-equivalent for Mg²⁺/dNTP. Hairpin and dimer ΔG are screening estimates — confirm critical designs with a dedicated secondary-structure tool. A 3′-end dimer/hairpin is the most likely to disrupt PCR.
A whole set of oligos, every pair
One oligo against itself is the first question; a multiplex panel or an inherited primer list needs every PAIR checked too. This screens up to 384 oligos for cross-dimers, flags duplicate sequences by name, reports the Tm spread across the set, and holds a 3′-end pairing to a stricter threshold than one that only sequesters a primer internally.
0 record(s) pasted — FASTA headers become the names; one sequence per line gets named oligo_1, oligo_2, …
A pairing that occupies an oligo's 3′ end counts as a conflict at a weaker ΔG than one that only sequesters primer internally, because that end is where extension starts. Defaults are −6 and −9.
Paste at least two oligos to screen the pool. For one oligo's own hairpin and self-dimer, use the Oligo Analyzer.
The full folding model, for one oligo or two
The nearest-neighbor screen above finds the most stable pairing. When you want the actual minimum-free-energy structure — the loops, the bulges, the dot-bracket — this folds one oligo or two together with ViennaRNA’s published loop model at your temperature and salt.
One oligo — folding it alone AND against a copy of itself, both from one run.
A whole multiplex panel, not just its oligos
The screens above judge oligos against each other. A multiplex panel also has to agree on ONE annealing temperature and produce amplicons a gel can tell apart, and a pair that dimerises is only one of the ways it fails. This checks the panel as a panel: Tm spread across the targets, product sizes that resolve, and the pairs that cannot go in the same tube at all.
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 says 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 worry about.
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 is worth 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.
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