Variant to Construct — Mutagenesis and Genotyping Primers from One Variant

One order table. For a single-base substitution with enough flanking sequence that is five oligos: a forward and reverse mutagenic primer that install the change, two allele-specific forward primers carrying the standard KASP universal tails (FAM for the reference allele, HEX for the alternate), and one common reverse primer. Each row carries its sequence as ordered, its length, its Tm, and the salt conditions and basis that Tm was computed under. You also get the full edited sequence, the reference context around the edit, and the recommended annealing temperature for the mutagenesis PCR.

The single-step tools each answer one question and each need you to hand them a sequence and a coordinate you have already worked out. This page starts one step earlier — at the variant identifier — and ends one step later, with both halves in the same table under one naming scheme. If you only need one half, the Mutagenesis Primer Designer (/tools/site-directed-mutagenesis) and the KASP / ARMS Primer Designer (/tools/kasp-primers) are the direct routes.

Not an rsID — the network mode parses HGVS "c." descriptions only, so convert the rsID to one (dbSNP lists the HGVS form) first. A VCF-style anchored allele pair is handled: both alleles carrying a shared flanking base ("T"/"TGGATCC", or "TG"/"AG") is trimmed to its minimal form before anything is designed, and the trimmed coordinates are reported, so an anchored SNV is designed as the substitution it actually is rather than as a delins.

KASP/ARMS discriminates two alleles by a 3'-terminal base that matches one and mismatches the other, and only a single-base substitution has such a base. So for an insertion, deletion, MNV or delins the genotyping half comes back as a named omission that says so and names the readout that does work for your variant instead — amplicon sequencing, a probe-based assay, or a sized flanking PCR when the two alleles differ by enough base pairs to resolve. The mutagenesis half still runs: every class goes through the same interval-replacement design.

A mutagenic primer needs a template-binding arm of at least 10 bp on each side of the change. An allele-specific genotyping primer ends on the variant base and its shortest usable core is 18 nt, so it needs at least 17 bp upstream, plus enough sequence downstream for the common reverse primer to sit inside your amplicon window (60-150 bp by default). Too small a window is reported as a window-too-small omission that says the variant is designable and the reference is not, rather than as "this cannot be done" — supply more flanking sequence, or a larger flank in the HGVS mode.

No, and this is the limitation most likely to bite you. There is no BLAST and no off-target search of any kind: every primer is designed against the reference you supplied and nothing else, so an oligo that also binds three other places in your plasmid, genome or transcriptome passes every check on this page. The gate says this itself, in its own list of things it does not verify, alongside oligo secondary structure and cross-dimerisation — put the ordered oligos through the Oligo Analyzer (/tools/oligo-analyzer), which scores hairpins, self-dimers and cross-dimers, before you order them.

No — the two stages' Tms are not on the same scale, and the table says which is which on every row. The mutagenesis rows are salt-corrected nearest-neighbour Tms of the whole primer; the genotyping rows are the untailed allele-specific core's Tm at a 1 M Na+ reference state, which for the same oligo runs several degrees higher. Neither is recomputed here, because reporting two different Tms for one oligo is how a plan stops being trustworthy. Put the whole table on one basis with the Oligo Analyzer (/tools/oligo-analyzer) if you need to compare them.

It refuses, quoting the bases actually found at that position and the sequence around them, whenever the reference allele you stated is not what your sequence reads there — the usual cause being a coordinate that is right for a different isoform, assembly or strand. That matters because a wrong coordinate does not fail loudly on its own: it produces a perfectly valid-looking primer set for the wrong base. What the check cannot tell you is that the sequence you supplied is the molecule you think you are holding; a variant whose allele happens to match at the wrong coordinate still passes.