PCR primer design tool

End-to-end primer and probe design for standard/inverse PCR, multiplex, quantitative fluorescence assays (TaqMan/MGB), bisulfite PCR, and Recombinase Polymerase Amplification (RPA).

Supports SNP and InDel genotyping with validated primer sets for qPCR and rapid isothermal RPA.

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 sequence from NCBI by accession — chromosomes (NC_), mRNA (NM_), RefSeq contigs

NCBI Accession:

Upload local FASTA file — plain FASTA, multi-FASTA, or sequences

Upload FASTA:

Primer Design Options

Length range (nt)

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Tm range (°C)

–

PCR product size (bp)

–

3′-end pattern (5′-3′)

Forward tail (5′-3′)

Reverse tail (5′-3′)

Non-specific priming control

Overlapping primers

Multiplex PCR

Inverse PCR (circular)

MGB-probe assay

TaqMan-probe assay

C→T bisulfite conversion

Recombinase Polymerase Amplification (RPA)

Sequences use standard IUB/IUPAC codes: B=CGT, D=AGT, H=ACT, K=GT, M=AC, N=ACGT, R=AG, S=GC, V=ACG, W=AT, Y=CT.
Region marking: Use [ and ] for Forward/Reverse primer regions; /.../ to exclude
RPA mode: Generates 30–38 nt primers for 37–42°C, short amplicons (100–200 bp)
Multiplex: Balances primer sets to minimize cross-dimers and ΔG conflicts
3′-end pattern: Use N for any, or specify patterns like WSS.
Linguistic Complexity (LC%) measures sequence vocabulary richness; 100% = maximum diversity.

Output: Export: Select all (Ctrl+A), copy (Ctrl+C), paste (Ctrl+V)

Quick Help for PCR Primer Design

Paste/upload FASTA or use NCBI/Ensembl retrieval (rsIDs + species) before pressing Generate.
Constrain primer search with [ ... ] and exclude regions with /.../ when needed.

Help: PCR tool · Help: Troubleshooting