MEDICAL GENETICS / RESEARCH WORKSPACE

PCR primer designer.

From a DNA sequence to a clear primer shortlist.
Less form-filling. More context for your next experiment.

Powered by Primer3Thermodynamic design · v2.6.1Research use

01 / TEMPLATE

Start with your sequence

Single DNA template

Upload FASTA

Waiting for a sequence120–20,000 bases

Paste plain DNA or one FASTA record. Spaces and line breaks are ignored. N marks an unknown base; primers will avoid it. Use a linear template or a linearized region of a circular template.

Project label optional · stays in this browser

Use public or synthetic DNA, without patient information. Clicking Design sends the sequence over HTTPS to our server for calculation. Human specificity checks run on our server. Primer sequences, product fingerprints and checking results are held privately for up to 15 minutes, then deleted; your full template is not saved. How it works

02 / DESIGN

What are you designing?

Standard PCRA general starting point150–600 bp Short ampliconFor example, a qPCR template80–200 bp Custom rangeSet your product length70–2,000 bp

Product minimum bpProduct maximum bpPairs to return

Every preset is editable. Product length includes both primer-binding regions.

Amplify a specific regionPlace the primers on either side of a target.

Fine-tune the designPrimer length, Tm, reaction conditions & exclusions

Primer length & melting temperature

Minimum length ntPreferred length ntMaximum length nt

Minimum Tm °CPreferred Tm °CMaximum Tm °C

Maximum pair ΔTm °CMinimum GC %Maximum GC %

Match your reaction conditions

These concentrations affect the predictions. Tm is not a recommended annealing temperature.

Monovalent salt mMMagnesium mMTotal dNTPs mM · sum of all fourAnnealing oligo concentration nM · Primer3 DNA_CONC

Regions to avoid

Excluded primer-binding regions optional

Use up to 10 start–end ranges in 1-based, inclusive coordinates. Products may span these regions; primers cannot bind within them.

Reject primer 3′ ends on lowercase bases

HUMAN REFERENCE CHECK

Look beyond your template

On this site

Automatically check every candidate pairRuns after design. Keep comparing pairs while the search continues. Human reference coverage

Adjust search thresholds +

Total mismatches maximum per primerLast 5 bases maximum mismatchesLargest product bp · includes both primers

Full-length, ungapped matches only. Increasing the mismatch limits includes more candidate binding sites. Products require inward-facing, non-overlapping primers. Insertions, deletions, population variants and circular-origin products are not covered.

GRCh38.p14 and its RefSeq RNA annotation. Searches stay on this server and usually take seconds to a few minutes. This is a bounded prediction, followed by experimental validation.

Usually a few seconds

METHODS & SCOPE

Clear about what is checked.

Version 1.1

Established design engine

Primer3 2.6.1 selects and ranks pairs using nearest-neighbor Tm calculations, SantaLucia salt correction and thermodynamic hairpin/dimer filters. Candidate coordinates and primer orientations are verified against your template.

We also count exact binding sites on both strands of your supplied template. This is a limited check, not a genome-wide specificity search.

Make the scope fit the experiment

Optional automatic specificity screening uses Bowtie 1.3.1 against locally hosted human GRCh38.p14 genome and RefSeq RNA references. It predicts inward-facing products, including same-primer products, under the visible mismatch and size limits. No SNP database, exon-junction annotation, probe design or multiplex panel analysis is included. The short-amplicon preset does not establish qPCR efficiency; transcript results must be interpreted in the context of the assay. Research use only; this is not a validated clinical diagnostic assay designer.

The sequence workspace is sandboxed separately from the advertising page. Your full template and project label are not saved by the tool. Specificity jobs temporarily hold primers, expected-product fingerprints and results in private server storage for up to 15 minutes. No sequences are sent to external checking services. Export anything you want to keep. External links open without automatically sending your sequence.

Primer3 methods ↗Engine source & GPL licence ↗Bowtie search methods ↗Human reference assembly ↗Independent check: NCBI Primer-BLAST ↗IDT OligoAnalyzer ↗

Primer3: Untergasser et al., 2012, Nucleic Acids Research 40(15):e115. Independent Medical Genetics workspace; no affiliation with the linked providers is implied. Advertising access does not change the design criteria or rankings.