PCR primer design tool
A PCR primer design tool finds pairs of short oligonucleotides that anneal on either side of a target region and amplify it cleanly: the right melting temperature, matched between the pair, no secondary structure, and a single binding site on the template. Talindrew's designer works on any selected region of an open sequence: it enumerates every candidate on each side, scores each on Tm, GC content, length, 3′ GC clamp, homopolymer runs, hairpins, self-dimers and how many places it binds, then pairs the best survivors and scores the pair on ΔTm, cross-dimers and product size. The list re-ranks as you move the constraints, every Tm is the same nearest-neighbour value as the melting temperature calculator, and finished primers are saved with the sequence, drawn on the map and exportable as CSV or FASTA.
Specifications
| Search | Every candidate on each flank of the selected region, then pairwise; runs client-side in milliseconds |
|---|---|
| Per-primer scoring | Tm, GC content, length, 3′ GC clamp, homopolymer runs, hairpins, self-dimers, 3′-anchored dimers, binding-site count |
| Pair scoring | ΔTm between primers, cross-dimers, product size |
| Default constraints | 18–30 nt (20 optimal), Tm 55–68 °C (60 optimal), GC 40–60 %, ΔTm ≤ 3 °C, GC clamp required, homopolymer ≤ 4, 40 nt search flank; all adjustable |
| Tm model | Nearest-neighbour thermodynamics with salt and oligo concentration corrections: shared with the Melting Temperature calculator |
| Binding-site search | Both strands, exact 3′ anchor plus a mismatch budget; amplicon prediction including products across the origin of a circular template |
| 5′ overhang builder | 13 restriction sites (EcoRI, BamHI, HindIII, XhoI, SalI, NotI, NdeI, XbaI, KpnI, SacI, NcoI, BsaI, BsmBI) with guard bases; Kozak; T7 promoter; His₆ tag; Gibson/HiFi homology arm; custom text |
| Tm reporting | Annealing-region Tm and full-oligo Tm shown separately |
| Primer library | Saved per sequence; import from FASTA or a pasted list; export CSV or FASTA; open in IDT OligoAnalyzer; exported as primer_bind features |
| Templates | Linear or circular DNA; GenBank, FASTA, EMBL, ApE, SnapGene .dna import |
| Price | Free |
How are the primers scored?
Scoring is a penalty, so a pair with penalty zero hits every optimum exactly and the list is sorted with the lowest penalty first. Each primer is charged for its distance from the optimal Tm and length, heavily for falling outside the allowed Tm or GC window, for a weak or over-strong 3′ GC clamp, for runs of identical bases, for hairpins and for self-complementarity: especially at the 3′ end, where a dimer becomes a self-priming artefact. A primer that binds the template in more than one place is charged again, because a second site means a second product. The pair then pays for the difference between the two Tm values, for cross-dimers between forward and reverse, and for a product outside the size range you asked for.
Why are two Tm values reported?
A cloning primer has two parts: the 3′ annealing region that binds the template, and a 5′ tail, a restriction site, a tag, a Gibson arm, that does not bind in the first cycles. The annealing temperature of the reaction is governed by the annealing region alone, so that is the Tm the designer optimises and reports first. The full-oligo Tm is shown beside it because it is what the tail-containing primer will do once the tail has been incorporated into the product, and because it is the number a vendor's oligo analyser will print. Using the full-oligo Tm to set the annealing temperature is one of the more common reasons a cloning PCR fails.
Checking where a primer binds
Before a primer is accepted the template is searched on both strands for every place the 3′ end could anchor, allowing a small number of mismatches further 5′. Predicted amplicons are listed with their size and position, including products that span the origin of a circular plasmid, which a linear search silently misses.
- One binding site per primer and one predicted amplicon is the goal.
- A second site with the same orientation produces a second band; a second site in the opposite orientation can produce a primer-dimer-like short product.
- Primers imported from a list or a FASTA file are located on the template automatically and checked the same way.
Adding restriction sites, tags and Gibson arms
The overhang builder prepends a 5′ tail to either primer. Restriction sites come with the guard bases the enzyme needs to cut efficiently near a DNA end; Kozak, T7 promoter and His₆ presets are placed in frame where that matters; a Gibson or HiFi arm takes its homology from the sequence you are joining to. Custom text is allowed for anything else. The annealing Tm does not change when a tail is added: the full-oligo Tm does, and both are shown.
Frequently asked questions
Is the primer design tool free?
Yes. Primer design runs in the browser as part of the free Talindrew workbench. Open or paste a sequence, select the region to amplify, and the designer returns ranked primer pairs immediately. No licence and nothing to install.
Which Tm calculation does it use?
Nearest-neighbour thermodynamics with salt and oligo concentration corrections, rather than a GC-percentage rule. It is the same function the Melting Temperature calculator uses, so a primer's Tm in the designer and in the calculator are always identical. Differences of a few degrees against another vendor's tool usually come from a different model or different assumed reaction conditions, not from an error.
Can it design primers with restriction sites or Gibson overhangs?
Yes. The 5′ overhang builder adds any of thirteen common restriction sites with guard bases, a Kozak sequence, a T7 promoter, a His₆ tag, a Gibson or HiFi homology arm taken from the sequence you are joining to, or custom text. The annealing Tm and the full-oligo Tm are reported separately, because only the first sets the reaction's annealing temperature.
Can I import primers I already have?
Yes. Paste a list of oligos or import a FASTA file and each primer is located on the template automatically, analysed for Tm, GC, hairpins and dimers, and added to the sequence's primer library. Export the library as CSV or FASTA, or send a primer to IDT's OligoAnalyzer for a second opinion.
Does it design primers for site-directed mutagenesis?
Mutagenic primers are designed by the site-directed mutagenesis cloning wizard, which produces QuikChange-style and NEB Q5 SDM primers for substitutions, insertions and deletions along with the protocol. The general primer designer described here is for amplifying a region.
Related
Try it on your own sequence.
Free to use. No download required. Works in any modern browser.