In-Silico PCR Tool
Simulate a PCR on your own template — see every product a primer pair can give, with mismatches and a specificity verdict.
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Allowing mismatches finds near-cognate sites — that is what reveals extra bands on a gel.
A template plus both primers are required.
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What it does
In-silico PCR answers a question that gel images answer too late: what will this primer pair actually amplify on this template? Instead of assuming one product, the tool enumerates every forward/reverse site combination within your size window and reports each amplicon with its coordinates, length and mismatch count.
How it works
Each primer is scanned against the template; the reverse primer is matched as its reverse complement on the top strand. Every forward site is paired with every downstream reverse site to form a product. Products where both primers match perfectly are the expected amplicons; the rest are potential extra bands. By default the 3′ terminal five bases must match, because a mismatch there usually kills extension.
Worked example
A 22 nt forward and 24 nt reverse primer on a 900 bp plasmid give one 524 bp perfect product and no others — the verdict is 'specific'. Allow two mismatches and a second 1.7 kb product may appear, which is the band you would otherwise blame on the polymerase.
When to use it
Use it to check a new primer pair before ordering, to explain unexpected bands after a reaction, and to confirm that a primer pair does not amplify the empty vector. It searches only the template you give it — for genome-wide specificity you still need a BLAST against the organism.
FAQ
- Is this the same as Primer-BLAST?
- No. Primer-BLAST searches an organism's genome database; this tool searches only the template you paste. It answers 'what happens in this tube', not 'what happens in this genome'.
- Why do I see products with mismatches?
- Because primers bind imperfectly too, especially at low annealing temperature. Those are the extra bands you would see on a gel.
- What does the 3' match requirement do?
- It enforces that the last five bases of the primer anneal exactly. Extension starts from the 3′ end, so a mismatch there usually prevents amplification entirely.
- Should my plasmid template be circular?
- Yes for an intact plasmid — a primer pair can amplify across the origin. Choose linear for a PCR product or a linearised fragment.