Genotyping Assay Design

Every design tool on this site in one workspace, as tabs: PCR and RPA primers, TaqMan / MGB / beacon / UniQ qPCR probes, castPCR, KASP, SNaPshot, MLPA, STR / SSR, a multiplex tiling panel, LAMP, Gibson assembly and in silico PCR. One input serves them all — pasted sequence, an Ensembl variant by rsID, an NCBI accession or an uploaded FASTA — so one target can go through several chemistries without re-entering it, each tool keeping its own design settings.

General-purpose primer design engine covering standard, inverse, multiplex, TaqMan, MGB-probe, bisulfite and RPA assays. Quantitative fluorescent PCR (QF-PCR) mode generates STR-flanking primers for dosage analysis and CNV genotyping.

Design TaqMan, MGB, Molecular Beacon and UniQ probes for quantitative allelic discrimination. Generates allele-specific probe pairs with matched Tms and minimal cross-reactivity, ready for 5′-nuclease, hairpin-based or strand-displacement detection, with multiplex mode for running multiple loci together.

Consensus PCR primers designed from a multiple sequence alignment of two or more sequences. Primers anchor on conserved regions every 3′-end is fully conserved across all inputs, with optional IUPAC-degenerate 5′-ends.

Design flanking primers for microsatellite genotyping by capillary-electrophoresis fragment sizing, with economical universal tail fluorescent labelling and automatic multiplex dye/size assignment. Pairs with the Repeats microsatellite finder.

Design allele-specific primers for KASP, PACE, ASQ and generic AS-PCR assays. Supports biallelic SNPs, InDels and complex haplotypes with automatic tail selection and Tm balancing. An allele-sizing mode trades the fluorophore for a gel: 5′-tails space the allele products into a ladder, so the genotype is read off band length with no reader at all.

Detect and quantify rare somatic mutations down to 0.1% mutant allele frequency. Designs a competitive triplet allele-specific primer (ASP) + allele-specific TaqMan/MGB probe + common counter-direction primer in both forward and reverse orientations, with multiplex mode for running multiple targets in one reaction.

Design single-base extension (SBE) primers for multiplex SNP and InDel genotyping on capillary electrophoresis platforms (Applied Biosystems SNaPshot). Also supports BAC fingerprinting and CpG methylation analysis by minisequencing.

Ligation-dependent half-probes (LPO + RPO) for copy-number (CNV), SNP and InDel detection read by capillary electrophoresis or, for up to 1000 targets, by NGS (digitalMLPA).

Toehold-mediated strand-displacement exchange probes for high-specificity single-nucleotide discrimination. The near-thermoneutral design maximises allele discrimination, with an enzyme-free, isothermal fluorescence readout on a simple plate reader.

Cas9 and Cas12a guides with activity and specificity scoring, an off-target search across the sequences you supply, HDR/ssODN donors carrying a silent PAM-blocking change, the primers that screen the edit, prime-editing pegRNAs with a Tm-matched PBS, and base-editing windows with bystander prediction.

Collateral-cleavage detection assays: Cas12a and Cas13a crRNAs with allele-specific synthetic-mismatch discrimination, RPA pre-amplification primers with a T7 promoter where the enzyme needs a transcript, and the quenched ssDNA/ssRNA reporter for fluorescence or lateral flow.

Polymerase cycling assembly: a target sequence becomes the set of overlapping oligos that builds it, with the junctions placed so every overlap melts at the same temperature, end primers for the second round of cycling, and long constructs split into blocks to be assembled separately and joined.