Primer map
Shows where each primer anneals on a template, on both strands, with the product sizes.
Shows where each primer anneals on a template, on both strands, with the product sizes.
Paste the template and a list of primers, and every primer is searched in both orientations at once. A primer whose own sequence is found on the plus strand is a forward primer at that position; a primer whose reverse complement is found on the plus strand anneals to the minus strand and is a reverse primer. Positions are always given on the plus strand of the template as you pasted it, counted from base 1, both ends included.
All IUPAC codes are understood on both sides, so degenerate primers and templates with N work without any extra setting. Two bases match when the sets of bases they stand for overlap: G matches S, and N matches anything.
Raise mismatches allowed to find sites that are not perfect. Mismatches are allowed anywhere in the primer except in the last three bases at the 3 prime end, which must pair exactly. That is where the polymerase starts, and a mismatch there blocks extension even when the rest of the primer is fully annealed. The same rule is what makes allele-specific PCR work.
Because the 3 prime base is checked first, a 100 kb template with a dozen primers is scanned in a fraction of a second and nothing freezes.
A forward site and a reverse site make a product when they face each other: the forward primer upstream, the reverse primer downstream. The product runs from the 5 prime base of the forward primer to the 5 prime base of the reverse primer, and both primers are part of it.
Pairs further apart than the longest product you set are left out, which keeps a long template from producing a table of pairings no polymerase would ever make. For melting temperatures of the primers themselves see the Tm calculator, for self and cross annealing the primer dimer checker, and to cut the product sequence out of the template the in silico PCR tool.
The built-in example is a 660 bp template with three primers: F1, Fnest and R1. At the default setting of 0 mismatches the tool reports 3 binding sites: F1 at 60 to 79 on the plus strand, Fnest at 200 to 219 on the plus strand, and R1 at 520 to 539 on the minus strand.
Two of the three pairings face each other, so 2 products are listed. F1 with R1 gives 539 − 60 + 1 = 480 bp, and the nested pair Fnest with R1 gives 539 − 200 + 1 =340 bp. F1 and Fnest point the same way, so they make no product.
Extra sites are the usual cause of a smeared or multi-band gel. Look first at sites whose mismatches are all near the 5 prime end, since those anneal almost as well as a perfect match. If two such sites face each other within a few kilobases, the product table will show the spurious band you are seeing. Shifting a primer by a few bases, or lengthening it at the 3 prime end, is usually enough. To search a template for any motif rather than a primer, usepattern find, and for a tolerant search with a free number of differences use fuzzy search.
Paste the template sequence, then paste the primers one per line with an optional name in front of the sequence. Every primer is searched in both orientations, and each binding site is listed with its strand, its position on the plus strand and the number of mismatches. Pairs that point towards each other are reported as PCR products with their size.
Polymerase extends from the 3 prime hydroxyl, so a base pair missing at the very last positions stops extension even when the rest of the primer is annealed. The search therefore allows mismatches anywhere except in the last three bases at the 3 prime end, which is the rule used when designing allele-specific and mismatch-tolerant primers.
Positions are always counted on the plus strand of the template you pasted, from base 1. A reverse primer binds the minus strand, so its 5 prime end sits at the higher position and its 3 prime end at the lower one. The arrow on the map points in the direction the polymerase extends.
Yes. All IUPAC codes are understood in the primer and in the template: R is A or G, Y is C or T, N is any base, and so on. A degenerate position counts as a match when the two sets of bases overlap, so a primer with three N positions is not penalised for them.
Either it is short, or it is degenerate, or the template contains a repeat. Sites with mismatches outside the 3 prime end are real priming risks in a PCR, which is why they are listed. Set mismatches allowed back to 0 to see only perfect sites, and check unexpected pairs in the product table for spurious amplicons.