Primer Checker
Test a primer for hairpins and dimers, scan a template for off-target sites, and list every product a primer pair could amplify.
Fill this in to test the two primers against each other for cross-dimers.
Only needed if you want off-target binding sites and mismatch counts.
Treat the template as circular, so binding sites and products that span the origin are included.
Learn more
What it does
A primer that looks fine as a sequence can still fail at the bench. Two problems account for most of those failures: secondary structure that stops the primer annealing, and non-specific binding that sends the reaction down the wrong template. This tool checks for both before you spend money on synthesis.
How it works
Four independent checks run on the sequence. Hairpin detection looks for a stem of four or more complementary bases closed by a 3–8 nt loop, reported with the stem and loop lengths. Dimer detection looks for a complementary 3' overlap of four or more bases between your primer and its partner, which is the configuration that blocks polymerase extension. When a template is supplied, the primer and its reverse complement are scanned across every position, including the circular wrap-around, counting mismatches and tracking separately how many fall in the 3' terminal five bases. A perfect match at one position plus a second site with one or two mismatches is the classic signature of a primer that will produce a background band. The fifth check works on the pair rather than on a single primer: both primers are annealed across every position of the template, and every combination that points at itself is enumerated as a candidate product. One perfect product means a clean reaction, a second product built from mismatched sites is the in-silico version of an extra band, and more than one perfect product means the template cannot tell your pair apart.
Worked example
Load the sample: the primer 5'-CACGCACCTAACAGTCTAAG-3' against its partner 5'-GTAACTCGCTAGGTTGGGTA-3' and the 750 bp template. Both primers report a Tm of 59.5 °C with 50% GC, no hairpin, and no 3' overlap with each other. Against the template the forward primer has exactly one perfect-match site at position 78 and the reverse primer exactly one at position 401, which the pair turns into a single 343 bp product with no competing site — the signature of a clean, specific pair.
When to use it
Run a pair through it before you order, especially when primers were designed elsewhere or handed down in a lab protocol. It is also the fastest way to diagnose a PCR that produces extra bands: set the mismatch tolerance to 2 and see whether the primer has a second binding site. The same check catches dimers that only appear at high primer concentrations.
FAQ
- What does a primer check actually test?
- Hairpin structures (a stem of four or more complementary bases with a 3–8 nt loop), primer dimers (a complementary 3' overlap of four or more bases between two primers) and, when you supply a template, every binding site within the mismatch tolerance you set — plus every product the pair could amplify once both primers are filled in.
- Which is worse, a hairpin or a primer dimer?
- A 3'-end dimer is usually worse. A hairpin can unfold during annealing, but a 3'-end dimer blocks the polymerase from extending directly, and the problem gets worse as primer concentration rises.
- What mismatch setting should I use?
- Two is the practical default: it catches most off-target products while keeping the list readable. Use 0 to find the intended binding site only, or 3 to be deliberately pessimistic about specificity.
- If the tool says my primer is fine, is it guaranteed to work?
- No. These checks cover secondary structure and, on the template you paste, both binding sites and the products the pair could amplify — including products built from mismatched sites, which is how a second band appears. They cannot search a whole genome or a sequence database, cannot predict template secondary structure or GC-rich amplification problems, and those genomic off-targets need a BLAST search against the actual genome.