PCR primer calculator

A common approximation formula for a single-stranded DNA oligo with a 5'-OH end (nA, nT, nC, nG being the count of each base). This is not an exact monoisotopic mass, and it is only computed for a primer with no ambiguous base at all (see the Limits section).

The polymerase extends the primer from its 3' end. Complementarity (with the other primer, or with itself) that touches this end can be extended and prime a spurious reaction; purely internal complementarity, further upstream, is markedly less problematic. That is why this tool always distinguishes global complementarity (the best alignment, wherever it sits) from complementarity at a 3' end (weighted more heavily in the risk assessment).

GC clamp

· presence of a G or C base at the terminal 3' position, generally considered favorable to annealing stability. A common design rule of thumb, not a guarantee.

Tm compatibility

· the two primers of a pair work better with close melting temperatures (a difference of a few degrees is commonly tolerated): a large gap favors preferential amplification of a single strand.

Heterodimer

· an unwanted pairing between the two primers themselves, consuming them instead of the target, more problematic still if it involves a 3' end.

Self-complementarity and hairpin

· a primer can bind to itself (dimer) or fold back on itself (hairpin structure), making it partially or fully unavailable to anneal to its target.

In PCR, two short primers flank the region to amplify: the forward primer anneals to the complementary strand in the 5'→3' direction of the region of interest, the reverse primer anneals to the opposite strand, oriented the other way. This tool accepts either one, or both. Analyzing a single primer is still useful to check its individual properties before even choosing its partner.

Melting temperature is computed with SantaLucia's (1998) nearest-neighbor model (explicit ΔH, ΔS. Effective concentration and salt correction, shared between both primers) whenever the sequence allows it, with the simple estimate (Wallace rule or GC-adjusted formula) always shown for comparison, never as a silent substitute. An ambiguous IUPAC base makes the thermodynamic calculation fail for that primer; if the other primer in the pair remains eligible, comparing the two Tm values directly would mix two different methods. The tool flags this explicitly rather than comparing silently. This model only accounts for monovalent cations (Na⁺/K⁺), not magnesium or dNTPs, and assumes two strands at equal concentration (see temperature-fusion-amorce for the full assumptions and their limits).

Complementarity between primers (dimers) and hairpin structure remain structural heuristics, distinct from the thermodynamic Tm above: this tool does not reproduce Primer3 or OligoAnalyzer on this point. Complementarity is detected by a gap-free scan; the hairpin structure, by a deliberately naive algorithm limited to primer-length sequences. One point matters: the « low / moderate / high risk » labels count consecutive matched base pairs and where the match sits (a 3' end weighs more, because the polymerase can extend it). They assess NO thermodynamic stability. Yet 4 G/C pairs are markedly more stable than 6 A/T pairs (ΔG°37 about −3.6 vs −2.9 kcal/mol): a GC-rich match can therefore be underrated, and an AT-rich one overrated. Tools that reason in ΔG (Primer3, OligoAnalyzer) remain the reference for settling a borderline case on dimers and hairpins. Every heuristic metric is flagged as such: these are risk indicators to interpret, never experimental validation results. An ambiguous IUPAC base produces a range (GC, simple Tm) or makes a calculation unavailable (molecular weight, thermodynamic Tm) rather than an invented central value. Finally, beyond 300 nucleotides (far longer than any real primer) the complementarity and hairpin searches are disabled and reported as such: their cost grows very fast with length, and they only make sense on a primer, not on a target sequence pasted by mistake.