castPCR assay design

castPCR (Competitive Allele-Specific TaqMan PCR) is a real-time PCR method for detecting and quantifying rare somatic mutations against a large excess of wild-type DNA. By combining allele-specific primers with MGB blocker oligonucleotides that suppress wild-type amplification, it routinely resolves mutant alleles down to 0.1% — sensitivity well below what Sanger sequencing can reach. Enable Multiplex castPCR to design a dimer-compatible castPCR set per locus, so several targets can be genotyped together in a single reaction.

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):

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

Human rsIDs:

Flank size (±bp):

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

Upload FASTA:

Primer Design Options

Length range (nt)

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Tm range (°C)

–

PCR product size (bp)

–

SNP at 3′-end of ASP

Allele-specific primer (ASP) tail (5′-3′)

Locus-specific primer (LSP) tail (5′-3′)

3′-end pattern (5′-3′)

Specificity

Non-specific priming control

Low complexity priming control

Allow overlapping primer positions

Multiplex

Multiplex castPCR

Designs one dimer-compatible castPCR set per locus so all targets can run together in a single reaction.

Methylation

C→T bisulfite conversion (methylation analysis)

Probe Format

MGB probe (12–21 nt)

TaqMan probe (18–30 nt)

Need details? Sequence markup, parameters, output columns and the suggested workflow are documented in Help → castPCR.
Exporting results: click inside the result area → Ctrl+A → Ctrl+C → paste into Excel.