Primer dimer and hairpin checker
Self-dimers, cross-dimers and hairpins with ΔG and an alignment for each. Paste one or two primers.
Self-dimers, cross-dimers and hairpins with ΔG and an alignment for each. Paste one or two primers.
A primer dimer forms when two primer molecules pair with each other instead of with the template. If the pairing leaves a 3′ end matched, the polymerase extends it, turning the pair into a short double-stranded product of roughly 40 to 100 base pairs that amplifies in every cycle. The result is the low band or smear at the bottom of a gel, weak or absent product, false positives in a no-template control, and inflated background in SYBR-based qPCR. Ahairpin is the same problem within one molecule: the primer folds back on itself, and a stable hairpin keeps the primer from binding the template at all.
Each primer is slid past the other, and past itself, in every register. At each register the tool finds runs of contiguous Watson–Crick pairs and scores them with the same nearest-neighbour parameters used by the Tm calculator, giving a free energy at 37 °C:
Hairpins are found by testing every stem and loop combination with a loop of at least three bases, adding a loop penalty that grows with loop size. The most stable structures of each kind are listed with an alignment so you can see exactly which bases pair. Only contiguous matches are scored: internal loops, bulges and mismatched neighbours are ignored, which makes this a fast screen rather than a full secondary-structure prediction.
| Result | What to do |
|---|---|
| No structure above the threshold | Nothing to do. Order the primers. |
| ΔG above −6, no 3′ pairing | Acceptable. Watch the no-template control. |
| ΔG −6 to −9, or any 3′ pairing | Reduce primer to about 100 nM, use hot start, raise the annealing temperature. |
| ΔG below −9, or 3 or more paired bases at the 3′ end | Redesign: shift the primer, or trim or extend the 3′ end. |
Redesign is the only certain fix, but several changes help. Use a hot-start polymerase so that no extension happens while the reaction is assembled at room temperature, and set up on ice if it is not hot start. Lower the primer concentration towards 100 nM, since dimer formation depends on primer meeting primer. Raise the annealing temperature, or run a gradient, because dimers are usually less stable than the true product. Increasing Mg²⁺ makes dimers worse, not better. In SYBR qPCR, a melt curve with a second, lower peak is the signature to look for.
As a rule of thumb, a 3′ dimer more stable than about −5 kcal/mol, or any dimer below about −9 kcal/mol, is worth redesigning. The 3′ end matters far more than the middle: a polymerase can only extend from a paired 3′ end, so three or four matched bases there cause more trouble than ten matched bases in the middle.
A self-dimer is one primer pairing with another copy of itself. A cross-dimer, or heterodimer, is the forward primer pairing with the reverse primer. A hairpin is one primer folding back on itself, with a stem of paired bases and a loop between them. All three take primer out of the reaction, and dimers can be amplified as a short product.
Because a dimer with paired 3′ ends is extended by the polymerase into a short double-stranded product, typically 40 to 100 bp, which then amplifies exponentially like any template. It competes for polymerase and dNTPs and can take over a reaction that has little real template, which is why dimers hurt most in low-copy and no-template controls.
Redesign is the reliable fix: shift the primer a few bases, or trim or extend the 3′ end so the complementary run is broken. Avoid G or C runs at the 3′ end and complementarity between the 3′ ends of the pair. If redesign is not possible, raise the annealing temperature, lower the primer concentration to about 100 nM, use a hot-start polymerase, and set the reaction up on ice.
It predicts base pairing and its free energy, which is the main driver, but not the whole reaction. It uses nearest-neighbour thermodynamics at 37 °C for contiguous matched stretches, without loops, bulges or mismatched neighbours, so it is a screen rather than a simulation. Treat a clean result as a good sign and a bad result as a reason to redesign.