SNaPshot® Multiplex SNP Genotyping

The SNaPshot® Multiplex System genotypes SNPs by single-base extension (SBE, minisequencing). An unlabeled SBE primer anneals with its 3′-end placed one nucleotide before the variant; a thermostable polymerase then incorporates a single fluorescently-labeled ddNTP opposite the SNP. The dye colour identifies the allele, the primer length identifies the locus after capillary electrophoresis.

Up to 10 SNPs can be interrogated in a single tube by giving each SBE primer a distinct length — typically shifted with a non-complementary poly-dT or poly-dGACT 5′-tail. The same chemistry supports InDel genotyping, BAC fingerprinting, and bisulfite-methylation analysis (C/T discrimination at CpG sites). This tool designs both the flanking PCR primers and the allele-interrogating SBE primers for every marker in the input, and cross-checks all oligos for primer-dimer compatibility across the multiplex.

Retrieve flanking sequence from Ensembl by rsID — validates species, fetches forward-strand context with allele bracket

Species:

rsIDs:

Enter Ensembl species id (lowercase, underscores). Custom species are stored in this browser.

Flank size (±bp):

Upload local FASTA file — plain FASTA, multi-FASTA, or sequences with [REF/ALT] brackets

Upload FASTA:

Cross-locus mPCR compatibility: every PCR primer pair listed for a given sequence has been pre-validated against the primers of all other sequences (no cross-dimers). You may freely combine any forward + reverse pair from one locus with any pair from any other locus in the same multiplex reaction — all listed pairs are interchangeable across sequences.

Primer Design Options

Length range (nt)

–

Tm range (°C)

–

PCR product size (bp)

–

3′-end pattern (5′-3′)

Forward primer tail (5′-3′)

Reverse primer tail (5′-3′)

Specificity

Mask repeats & low-complexity regions

Allow overlapping primer positions

Methylation

C→T bisulfite conversion (methylation analysis)

Sequence Format & Notation

SNP / InDel markup

Biallelic SNP: flank [A/G] flank — alleles separated by /.
Shorthand (IUPAC): flank [R] flank — R = A/G, Y = C/T, etc.
InDel: flank [ATCG/-] flank for an insertion vs. deletion.
Excluded region: wrap with forward slashes / .../ — primers and SBE probes avoid it.

IUPAC ambiguity codes

R=AG, Y=CT, S=GC, W=AT, K=GT, M=AC, B=CGT, D=AGT, H=ACT, V=ACG, N=ACGT

PCR primer 3′-end pattern

N — any nucleotide (no constraint).
W (A/T) and S (G/C) — composition codes for the last 2–3 bases.
Space-separated list: every pattern is tried and the best primer is kept.
Default sws ssw sww wss www favours a single G/C clamp without runs.

SBE primer design

3′-end of the SBE primer is fixed one nt before the SNP; the ddNTP incorporated identifies the allele.
Forward and reverse SBE primers are generated so the more specific orientation can be chosen.
Probe length 16–35 nt; extend with a 5′ poly-dT or poly-dGACT tail to shift mobility on CE.

Output columns

ID · Sequence · Length · Tm · CG% · LC% · YR%
For each assay set: PCR primer pair + SBE primer(s); the PCR row also reports Fragment size (bp) / Annealing Tm (°C).

Cross-locus combinatorial use

All listed PCR primer pairs are pre-checked for cross-dimers between sequences. Any forward + reverse pair from one locus can be freely combined with any pair from any other locus in a single mPCR — pairs are fully interchangeable across loci, so you can pick whichever pair has the best Tm/size for each target.

Exporting results: Click inside the result area → Select All Ctrl+A → Copy Ctrl+C → Paste into Excel or a text editor.

Quick-start guide

Enter sequence(s) — paste a multi-FASTA with each SNP marked as [REF/ALT] (e.g. …flank[C/T]flank…), upload a local FASTA, or retrieve flanking context by rsID from Ensembl.
Set parameters — PCR primer length / Tm / amplicon size, optional 3′-end composition pattern, and 5′ mobility-shift tails for the forward and reverse SBE primers (poly-dT or poly-dGACT). Tick Mask repeats to avoid low-complexity flanks; tick C→T conversion for bisulfite-methylation assays.
Press Generate — the Report tab lists every candidate PCR primer and SBE probe per locus; the Multiplex Assay Sets tab returns validated, dimer-free PCR + SBE combinations ready for ordering. Pairs from different sequences are pre-validated to be cross-locus compatible, so any F+R pair from one locus can be mixed with any pair from any other locus in the same mPCR.
Plan the multiplex — stagger SBE primer lengths by ≥6 nt (using the 5′ tails) so all loci resolve as distinct peaks on capillary electrophoresis.

Help: SNaPshot workflow · Help: Troubleshooting