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Primer Hairpin and Dimer Checker

Screen a primer or a primer pair for hairpins, self-dimers and cross-dimers by finding runs of complementary bases, and see whether a 3′ end takes part.

Formula

hairpin: a stem of n paired bases separated by a loop of 3 or more\text{hairpin: a stem of } n \text{ paired bases separated by a loop of 3 or more}
dimer: longest uninterrupted run of Watson-Crick pairs between two strands annealed antiparallel\text{dimer: longest uninterrupted run of Watson-Crick pairs between two strands annealed antiparallel}
stem\text{stem}
the paired bases of a hairpin, in base pairs
run\text{run}
consecutive complementary pairs between two primers
3′ run3'\ \mathrm{run}
a run that reaches the 3′ end of a primer, where a polymerase can extend it

How it works

A primer can fold back on itself into a hairpin, or pair with another copy of itself or with the other primer into a dimer. A polymerase can extend the 3′ end of a primer that is paired with another primer, which produces a short, efficiently amplified artefact, the primer-dimer, at the expense of the target.

The check looks for complementary runs by sliding the strands past each other. It reports the longest run, and separately the longest run that includes a 3′ end, which is the harmful case. It is simple sequence matching: it does not calculate free energy, so it cannot tell a stable GC-rich run from a weak AT-rich one of the same length.

Worked example

The M13 forward primer GTAAAACGACGGCCAGT alone and with the M13 reverse primer CAGGAAACAGCTATGAC.

  1. The longest self-complementary run in the forward primer is GGCC (4 base pairs).
  2. No stem of 4 or more with a loop of 3 or more exists, so there is no hairpin.
  3. The longest run between the two primers is 2 base pairs.

A 4-base self-dimer run, no hairpin and a 2-base cross-dimer run: nothing above the level usually of concern.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Only A-T and G-C pairs are counted; wobble pairs are not.
  • A hairpin needs a loop of at least 3 bases.
  • Sequence matching only, at no particular temperature or salt.

Common mistakes

  • Treating a short run as harmless regardless of whether it includes the 3′ end. A 3-base 3′ run can be enough to prime.
  • Relying on this tool alone for a difficult design. Use a thermodynamic calculator, and ultimately test the primers.
  • Forgetting the dimer of a primer with itself in a high-concentration reaction.

Related tools

Oligo and Primer AnalyzerLength, GC, Tm, weight and a design-guideline check for a primerOpen calculatorPrimer Pair AnalyzerTm match, GC, dimers and product size for a forward-reverse pairOpen calculatorPrimer Tm Calculator (Four Methods)Primer melting temperature by four accepted methods, side by sideOpen calculatorReverse Complement ToolReverse, complement or reverse-complement a sequenceOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.