primer_specificity

com.seqbench/workbench · Verify this server

Self-hosted e-PCR-style screen for off-target amplicons predicted by a primer pair against a small set of curated reference genomes (currently: E. coli K-12 MG1655, B. subtilis 168, human mitochondrion rCRS, Mycoplasma hyorhinis SK76 — see genomesChecked in the response for the exact list, and note that the nuclear human and mouse genomes are NOT covered). Amplicons are 1-based inclusive on the plus strand; a product across a circular genome's origin reports an end lower than its start and sets wraps: true. This checks background/host-genome specificity, NOT whether the primers hit your intended target — pair it with in_silico_pcr against your own template for that. Each off-target end reports its 3' ANCHOR — the primer's unbroken run of matched bases at the extending end — with that anchor's nearest-neighbour ΔG and a margin against the intended, fully matched reaction, so a site can be told apart by WHERE its mismatches fall rather than only how many there are: one mismatch at the 5' end leaves a site nearly as strong, and one at the 3' base leaves it unable to prime at all. Batchable over candidate REVERSE primers against one fixed forward primer (screen many candidates against a shared partner) — not independent primer-pair batching, which this tool doesn't support. A primer may carry a non-templated 5' tail (a restriction site, a Gibson arm, a tag): the screen looks for a 3'-anchored annealing region as well as a full-length match, so a tailed cloning primer is screened rather than silently matching nothing. Each end's `anchor` is the annealed run, which is the length that matters for extension, and `start`/`end` are measured on the ANNEALED footprints — the bases each primer actually pairs with on the genome — so `length` (the product, tails included) equals end - start + 1 only for untailed primers. Screening a TAILED primer without `intendedTemplate` inflates every margin by the tail's own free energy, because nothing about an oligo says where its non-templated part ends; `intended.basis` reports which footprint the margins rest on.