SNP/InDel Genotyping — TaqMan / MGB & Molecular beacon Probe Design

End-to-end primer and allele-discriminating probe design for quantitative fluorescence (qPCR) genotyping assays.

Read Instructions

Automated design of allele-discriminating probe–primer sets for SNP and InDel genotyping by quantitative PCR (qPCR) using dual-labelled probes.

Supports TaqMan (hydrolysis), MGB (minor groove binder) and Molecular beacon (hairpin-shaped) dual-labelled probes formats. Sequences can be fetched directly from Ensembl by rsID or loaded from a local FASTA file. All primers and probes are screened for Tm, hairpins, primer–dimer interactions, repeat masking, and linguistic complexity.

Retrieve flanking sequence from Ensembl by rsID — validates species, fetches forward-strand context with allele bracket
Enter Ensembl species id (lowercase, underscores). Custom species are stored in this browser.
Upload local FASTA file — plain FASTA, multi-FASTA, or sequences with [REF/ALT] brackets
Primer Design Options
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Specificity

Methylation

Probe Format

Sequence Format & Notation

SNP / InDel markup

  • Biallelic SNP: flank [A/G] / [R] flank
  • InDel: flank [ATCG/-] flank (deletion = -)
  • Tri-/tetra-allelic: [A/C/G/T] / [N]
  • Excluded region: / .../ — forward slashes

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

3′-end pattern codes

  • N — any nucleotide (no restriction)
  • W — weak (A or T) for better allele discrimination
  • S — strong (G or C)
  • Space-separated list: all patterns are tried, best result kept
  • Example: sws ssw sww wss www

Output columns

ID · Sequence · Length · Tm · CG% · LC% · YR%
Primer pairs also show: Fragment size (bp) / Annealing Tm (°C)

Exporting results: Click inside the result area → Select All Ctrl+A → Copy Ctrl+C → Paste into Excel or a text editor.
Quick-start guide
  1. Enter sequence — paste a FASTA with [REF/ALT], upload a file, or retrieve by rsID / accession.
  2. Set parameters — adjust Tm range, product size, probe format (TaqMan / MGB / Molecular beacon), and tick SNP/InDel mode.
  3. Press Generate Primers — results appear in the Report and PCR Primer Pairs tabs.
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