KASP / ARMS Allele-Specific Genotyping Primer Designer
Design KASP/ARMS SNP-genotyping primers — two allele-specific forward primers with FAM/HEX tails and an engineered ARMS secondary mismatch, plus a common reverse primer.
🔒 Nothing you paste is logged or stored
Give a SNP position and its two alleles on a target sequence, and get a complete KASP/ARMS genotyping assay: two allele-specific forward primers that differ only at the 3' terminal base (each matching one allele) carrying the standard KASP universal tails (FAM for allele A, HEX for allele B), a deliberately engineered second mismatch a few bases in from the 3' end whose destabilising strength is chosen to complement that primer's own natural allele mismatch — the classic ARMS trick for sharpening allele discrimination — and one common reverse primer placed downstream within your chosen amplicon-size window. It reuses SeqBench's nearest-neighbor Tm engine, so primer Tms are computed the same way as the rest of the site. This niche is served today mostly by ageing, crop-specific, or spreadsheet-based tools; this is a clean, free, browser-based option.
Position 18 or later — the allele-specific core needs ≥17 bp of template upstream of the SNP.
Working on a whole construct rather than one SNP assay? Open SeqStudio — the full editor, with live feature annotation, plasmid maps, restriction and primer panels, undo/redo, multi-document tabs and GenBank / SnapGene import and export.
How to use the KASP / ARMS Primer Designer tool
- 1Paste the target region with flanking sequence on both sides of the SNP — at least 17 bp upstream so the allele-specific core has template to build from (the SNP must sit at position 18 or later), plus 3' flanking sequence so a common reverse primer fits — then enter the SNP's 1-based position and its two alleles.
- 2Review the two allele-specific forward primers (FAM/HEX tail + core) — each shows its own natural 3' allele mismatch and the engineered ARMS secondary mismatch chosen to complement it — plus the common reverse primer with its amplicon size.
- 3Copy the three primers to order — then validate allele discrimination empirically, since it depends on your assay conditions.
Frequently asked questions
How do the allele-specific primers work?
Two forward primers share everything except their 3' terminal base, which matches one allele each. A polymerase extends efficiently only from a perfectly matched 3' end, so each primer amplifies preferentially from its own allele. Allele A's primer carries the FAM universal tail and allele B's the HEX tail, so the two alleles read out on different fluorescent channels; a single common reverse primer serves both.
What is the engineered secondary mismatch (ARMS)?
A single 3' terminal mismatch is often not discriminating enough on its own, so ARMS assays introduce a second, deliberate mismatch a few bases in from the 3' end. This tool places it at the third base from the 3' end and picks its identity so its destabilising strength complements that primer's own natural allele mismatch (a strong allele mismatch is paired with a weaker secondary mismatch, and vice versa). The two primers are handled independently, because they do not share a mismatch class: a forward primer anneals to the antisense strand, so its 3' base sits opposite the complement of the other allele. An A/C SNP, for instance, gives the A-primer a strong A:G mismatch but the C-primer only a weak C:T, so the two need opposite secondary mismatches. Classes are graded from the measured PCR yields in Kwok et al. 1990 (Nucleic Acids Res 18:999—1005): A:G, G:A and C:C cut yield ~100-fold, A:A ~20-fold, and the rest amplify efficiently. You can disable the secondary mismatch via the API.
How are Tm and the reverse primer chosen?
The allele-specific core (without the universal tail) is sized so its nearest-neighbor Tm is near your target (default 56 °C); the tail contributes additional Tm once incorporated in later cycles. The common reverse primer is searched downstream for an 18–24 nt window whose Tm best matches, within your amplicon-size range (default 60–150 bp).
Why is my SNP position rejected?
The allele-specific primers end on the SNP, so they are built entirely from template upstream of it. A SNP closer than 18 bp from the start of the sequence you pasted leaves too little to make a core from, and the design is refused rather than returned as a too-short primer. Re-paste the target with more sequence 5' of the SNP and shift the position accordingly.
Does it guarantee the assay will discriminate cleanly?
No. Allele discrimination in KASP/ARMS is empirical and condition-dependent (touchdown cycling, salt, template quality). This tool applies the established design rules and reports Tm/GC/mismatch strength transparently, but does not simulate the assay — validate with known control genotypes.
Is my sequence stored, and can I use this from code?
No. Your target sequence is used only to design the primers and is never logged or stored. The kasp_primer_design tool is available via the REST API and MCP server.
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