Check primer specificity against both strands of a host genome or custom reference. Compare possible PCR products by annealing temperature, consecutive 3' matches, product size, and gene overlap.
For host-background screening, choose the host genome instead and enter the intended plasmid amplicon size. That size compares host products; it does not load or scan the plasmid sequence. Use a separate custom-reference scan when you also want to check the plasmid itself.
Files must be uncompressed and no larger than 60 MB. All records combined must contain no more than 50,000,000 bases, including Ns. Custom references provide binding coordinates and product lengths; uploaded GenBank annotations are not currently used for gene labeling.
Your custom reference is read and scanned in your browser, not uploaded to our server or saved in an online account. Keep your original file if you need to scan it again. The bundled genome download is separate: selecting a strain retrieves its reference files from this site.
The input checks validate readable sequence and size limits, not strain identity or assembly quality. Confirm the source and assembly version yourself. No off-target found means none was found within the supplied sequence and selected search limits, not that your PCR is guaranteed to be specific.
See NCBI's nucleotide FASTA guidance for record structure. Primer Scan's A/C/G/T/N alphabet is narrower than the full IUPAC alphabet allowed by FASTA.
No indexing software or manual preprocessing is required. Built-in and custom references use the same search engine in a background browser worker. Each scan builds short sequence lookup keys from the primers, searches both orientations, and evaluates the candidate binding sites. Repeated thermodynamic calculations are cached within the scan.
Bundled genomes use verified two-bit DNA files. The scanner searches this compact representation directly; unknown N bases and record boundaries are preserved. This changes storage and search speed, not the binding criteria. Custom DNA, FASTA and GenBank inputs use the text-based search path and do not need conversion by the user.
Gene annotations are checked against the reference sequence and added after scanning. The scan does not assemble reads, join contigs, or remove repetitive DNA. Large or repetitive references and permissive settings can increase runtime or reach a resource limit; an incomplete scan is reported as an error.
Each predicted product lists its final length and the genes or RNA features it overlaps. Expand Gene context for locus tags, annotated products, gene direction, coding-sequence coverage, and the overlap of each primer with the gene. Gene context is added after the sequence scan; annotation loading does not change the predicted products or their binding assessment.
The library includes E. coli K-12 MG1655, DH5α, TOP10 and BL21(DE3); B. subtilis 168, WB800N and PY79; yeast S288C, CEN.PK113-7D and BY4742; and Aspergillus niger ATCC 1015 and NRRL3. Both DH5alpha and DH5α work in the strain search. Sequence-matched gene annotations are available for all except NRRL3. Custom references and NRRL3 provide binding coordinates and product lengths without gene labels.
CEN.PK113-7D and BY4742 use ScRAPdb gene models, verified against all 16 nuclear chromosomes and the source coding sequences. Source gene names and locus identifiers are retained. Slash-separated names indicate multiple candidate identities; they are not a resolved gene assignment. The seven bacterial strains and S288C use sequence-verified NCBI annotations. Source names, identifiers and retrieval or update dates appear in Gene context.
A. niger ATCC 1015 uses GCA_055697485.1 (2026): eight nuclear chromosomes, 35,617,925 bases and 10 unresolved N bases. Its GFF3 coordinates were checked against all 11,113 deposited CDS sequences. NRRL3 uses the 35,245,396-base JGI/Genozymes genome from the public CoGe copy. Its 15 scaffolds are scanned separately; products spanning missing centromere sequence cannot be predicted. This copy has no gene annotations. See the NRRL3 genome publication.
No annotated gene overlaps means the product falls outside the genes in the supplied annotation. Nearby genes are shown only as flanking context, not as part of that product. Annotation unavailable means gene context could not be assessed; it must not be interpreted as an intergenic product.
Entire annotated CDS included describes coordinate coverage, not a guarantee of an intact, expressed or functional protein. Genomic products can include introns. Non-binding primer tails contribute to final product length but are not assigned to reference genes.
In the results, an optional expected reference product length compares candidates in base pairs and percent without filtering them. This is separate from the plasmid-versus-host background check below. Summary and CSV export become available when the annotation lookup finishes or reports that annotations are unavailable.
Use Send results to Primer Scan below the results in Primer Designer or Sanger Primer Designer. Full primer sequences, names and pair assignments are retained, including cloning overhangs. No template DNA is transferred: select the host genome or supply your own reference in Primer Scan.
Transfers stay in this browser and expire after 30 minutes. Sanger designs with more than five pairs transfer only the first five, with a warning on both pages. Cross-pair checking stays off until selected. Sanger sequencing uses one primer per reaction; scanning its forward/reverse pairs assesses possible PCR products, not sequencing quality.
When checking a cloned gene by colony PCR, select the cloning or expression host's genome and enter the intended plasmid PCR product size for each primer pair. Use the amplicon length between your primers, including any non-binding tails, not the total plasmid size. The intended plasmid product does not need to exist in the scanned genome.
Entering an intended plasmid product size automatically flags reference products within plus or minus 10% of that size. For a 1,000 bp intended amplicon, candidates from 900 to 1,100 bp are included. Multiple nearby products are counted in the results; even one genomic candidate may interfere with an intended plasmid band. Leave the size blank to omit this comparison.
The fixed 10% window is a screening aid, not a validated gel-resolution cutoff. Products meeting the sequence or temperature screen, needing review, and below the screen are counted separately. No products are removed by this comparison, and there is no separate size-comparison setting.
Separate reactions use their own plasmid sizes. When cross-pair checking is enabled, cross-pair products are compared with every entered pair's plasmid size. Check the named pair in each flag. Entering a plasmid size does not add its sequence to the scan or verify that the plasmid will amplify.
If the amplicon size limits exclude any part of the comparison window, a warning asks you to widen the limits and rescan. No nearby genomic candidate is not proof of no background amplification: omitted sequences, indels, mismatches outside the selected limits, and unmodeled reaction conditions can affect the result.
Similar-sized PCR products may co-migrate, but size alone cannot establish whether bands will separate. Agarose concentration, fragment length, sample loading, and electrophoresis conditions matter. A product outside the chosen window is not guaranteed to be removable by gel purification. See Thermo Fisher's nucleic acid gel electrophoresis guide.
Primer Scan is an alternative to NCBI Primer-BLAST for checking existing primers against a selected reference. It does not design primers or search NCBI's sequence databases, and equivalent sensitivity has not been established. Use Primer Designer to design primers, then scan them against the relevant host reference.
Perfect matches use SantaLucia 1998 nearest-neighbor parameters with Owczarzy salt correction, shared with the PCR and Sanger designers. Isolated internal mismatches replace the affected stacks with experimental Allawi/SantaLucia and Peyret parameters; no fixed degrees-per-mismatch penalty is applied.
Each supported mismatch must have matched bases on both sides. Multiple mismatches must be separated by at least two matched bases. Terminal mismatches, closer clusters, and self-associating mismatched duplexes are unscored and retained for review when permitted by the search settings. Gapped alignments are not searched: binding sites requiring an insertion or deletion may be missed.
Mismatch Tm values are model estimates, not experimental measurements. The salt correction was developed for DNA duplexes and is an approximation when applied to mismatches; A.C mismatches are also pH-sensitive. Manufacturer buffers, additives, polymerase choice, and reaction kinetics can change experimental behavior.
Receiving primers: Primer Designer can send its generated pair here; Sanger Primer Designer can send up to five pairs. These transfers fill the primers and names, not a genome or template. Choose the host reference or supply custom DNA, confirm topology and scan settings, then run Scan primer specificity.
Manual input from newer designers: copy the full primer pairs from Assembly PCR, Gibson or Mutagenesis, then choose the relevant reference. Use the original PCR template to check a fragment reaction, the final construct to map the completed design, or the host reference for background candidates. Review 5′ tails against the reference actually being scanned.
Using results elsewhere: export the product CSV or copy the summary to keep coordinates, sizes and review reasons. There is no direct send-to-another-tool button. To inspect one template in Primer Binding Checker, supply its sequence and the relevant full pair manually. A reported host-product size is not the same as transferring that reference sequence.
When the main action rejects pasted sequence text, unsupported characters are highlighted in the field and the error message explains the problem. Correct the marked characters using the alphabet allowed by this tool, then run it again. The highlights update as you edit. A malformed FASTA or GenBank record may require a structural correction even when no individual character is marked.