SNaPshot single-base extension primer design

The SNaPshot® Multiplex System genotypes SNPs by single-base extension (SBE, minisequencing). An unlabelled SBE primer anneals with its 3′-end placed one nucleotide before the variant; a thermostable polymerase then incorporates a single fluorescently-labelled 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 bisulphite-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 checks the PCR primers of all loci against one another for primer-dimer compatibility across the multiplex.

Retrieve flanking sequence from NCBI by rsID — human dbSNP, fetches forward-strand context with allele bracket

Human rsIDs:

Flank size (±bp):

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)

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Tm range (°C)

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PCR product size (bp)

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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 bisulphite conversion (methylation analysis)

Need details? Sequence markup, parameters, output columns and the suggested workflow are documented in Help: SNaPshot workflow · Help: Troubleshooting