Primer Binding Checker Help

Useful Checks

• Add up to 10 primer pairs and check each against the same template. Primers from different pairs are not combined to predict cross-pair products.
• Use the primer map to compare the expected site with additional exact matches elsewhere in the entered template. This is not a genome-wide off-target search.
• Open the same template in Primer Designer to redesign a primer, or in Restriction Site Analyzer to inspect enzyme sites.
• The transfer buttons in Primer Designer and Sanger Primer Designer fill in the template and generated primers here.

How To Use

1. Paste or upload the template.
2. Choose linear or circular topology.
3. Enter primers in 5' to 3' direction.
4. Add more primer pairs if needed.
5. Click Check primer binding, then review each pair's map, exact matches and predicted products.
6. Changing calculation inputs clears the previous result. Run Check primer binding again after editing.

Understanding The Results

• The quick result reports products predicted from the exact-match rules, not experimentally confirmed amplification.
• Use Full-template context to locate binding sites on the original template and Primer pair summary for coordinates and product lengths. In Highlighted template view, blue marks forward binding, red marks reverse binding, black marks overlapping binding regions, and yellow marks non-binding 5' sequence. The reverse primer is drawn antiparallel, 3' to 5', to align it with the template. Enter and order both primers 5' to 3'.
• Unmatched 5' bases are labelled as overhangs. Check these against your design: a sequence error or difference from the template can also appear as an inferred tail.
• If known restriction sites are detected in the forward and reverse 5' overhangs and their cohesive ends are compatible, the pair details show a restriction-end warning.
• Additional matches suggest possible off-target binding. Extra PCR products also require suitable primer orientation, spacing and reaction conditions.
• The template amplicon size spans the selected binding sites. Circular whole-plasmid products can include repeated terminal template bases when the inferred binding regions overlap. These bases count toward the linear product length; the full-template map shows one reference copy. Final PCR product size also includes detected 5' overhang bases.
• An imported Primer Designer region selects the matching intended product when available; otherwise the summary uses the shortest compatible candidate.
• Pair details list alternative candidates, and the product-candidates TSV includes every compatible combination of the detected binding sites.
• Copy summary copies the text results. Choose SVG or PNG in Map format and click Download map to save the full-template map. Download product candidates TSV saves all compatible candidates, including alternatives to the selected product.

Read the template map and primer results

The full-template context retains the original coordinates. Choose a pair, or All pairs, and enable Show additional binding sites to inspect other detected sites. The initial map shows the first pair's selected sites and states how many additional sites are hidden. Solid direction symbols identify selected sites; hollow symbols identify additional sites. The primer pair summary gives the selected binding coordinates, template amplicon length, final product length and selection basis. All match counts, warnings and compatible products remain available in Pair details and the TSV.

On the circular map, coordinates increase clockwise from base 1. Colored arcs follow the actual binding intervals, direction symbols end at the true 3' coordinates, and leaders connect labels to their sites. Nonbinding 5' tails appear in the highlighted template view and Pair details; they do not have plasmid coordinates. The map retains a readable width on small screens. Reach it or a table with Tab, then use arrow keys to scroll.

Accepted Input Formats

• Template: raw DNA sequence, FASTA, or GenBank text with an ORIGIN sequence section.
• Primers: A/T/G/C DNA sequence, written 5' to 3'.
• FASTA headers and GenBank annotations are ignored. Multiple FASTA records are concatenated, so analyze separate templates one at a time.
• .dna / SnapGene binary files are not supported. Export as FASTA or GenBank.

Assumptions And Limitations

• The tool requires exact binding in the accepted 3' binding region. It does not allow mismatches, degenerate primer bases, or inosine.
• Minimum binding length can identify an exact 3' binding suffix while treating unmatched 5' sequence as an overhang; it is not mismatch tolerance.
• For each primer, matches are reported at its longest matching 3' suffix. Shorter binding matches and products with the primers in other orientations are not exhaustively screened. Product spans must contain both complete binding regions; an overlap alone does not establish a valid amplicon.
• Restriction enzyme identity is inferred from recognition sequences in detected 5' overhangs. Compatible-end warnings include Type IIS and degenerate-site enzymes only when the full cleavage region can be resolved from the overhang. Sites whose cuts extend into the binding region require review in Restriction Site Analyzer using the complete product sequence.
• The tool does not calculate primer Tm or thermodynamic binding strength.
• A result with no binding warnings does not guarantee PCR success.
• Use a separate database specificity search when you need to check sequences beyond the supplied template.
• The browser tool accepts sequence files smaller than 2 MB and templates up to 500,000 bp.

Example

Imported designs

Assembly, Gibson and Mutagenesis designers send the final construct and named primers here. All pairs share one template view. Original PCR templates are not checked. Transfers expire after 30 minutes and are removed after import.

Use this result with other tools

Direct destinations: Open in Primer Designer sends the current validated template and topology to Primer Designer to design a new pair. Open in Restriction Site Analyzer sends that template and topology to Restriction Site Analyzer to map recognition sites and plan a digest. These two buttons send the template, not the entered primer pairs or predicted PCR-product sequence.

Receiving a design: Primer Designer can send its template and designed pair. Assembly PCR, Gibson and Mutagenesis send the final construct and named pairs; Sanger sends its template and selected sequencing-primer pairs. Check the import notice, topology and pair names, then run Check primer binding. A final-construct check does not replace checks on the original fragment PCR templates.

Manual follow-up: copy the full primers into Primer Scan and select a host or custom reference to check beyond this one template. There is no direct send-to-Scanner button here. If you want to reuse a predicted product, copy that product specifically rather than using the input-template transfer buttons.

Sanger imports retain their original primer names beside the F1/R1, F2/R2 labels. Current Sanger designs contain up to 10 pairs and transfer the complete plan together. Assembly, Gibson and Mutagenesis designs can use selectable batches of up to 10 pairs; each import shows its range within the complete design. Older transfers without a batch range state imported and omitted counts. Sequence and primer handoffs expire after 30 minutes and can be imported once.

Correcting highlighted input

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.