Primerool

Primer & probe design for any organism: from a gene name to checked oligos in minutes.

Features · Quick start · How it works · Architecture · Development

Primerool fetches genes live from Ensembl or NCBI, lays them out as an interactive sequence map, and lets you place primers and probes by hand or have them designed for you. Every oligo is checked right away for Tm, GC content, hairpins and dimers by its own native thermodynamics engine, Strider. You don't need genome files, a database or an install: open it in a browser.

Any organism 51 preset species across animals, plants, bacteria, fungi, protists and viruses, plus any Ensembl species name. You can also paste an accession or a raw sequence.	Direct design Select bases, right-click and choose what to make. Drag a primer to move it, or drag its end to resize it. Everything is recomputed as you go.	One native engine Strider (native Rust nearest-neighbour + Mathews 2004 folding) picks and checks every oligo, with an IDT OligoAnalyzer Tm next to Strider's when you want a second opinion.
Every assay type General PCR, whole-gene (WGA), exon–exon junction (qRT-PCR), ARMS allele-specific, TaqMan and allele-detection probes.	Alignments MAFFT multiple alignment shown the classic way (numbered rows and a match line), plus primers designed in conserved regions.	Keeps your work Primer sets remembered per sequence, sessions saved to a file or autosaved, and light/dark themes with your own accent colour.

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Features

Find your gene

• Search by gene name (e.g. BRCA1, CSN2) or accession (e.g. NM_001443849.1). An accession is identified through NCBI BLAST and resolved to its gene.
• Ensembl REST or NCBI E-utilities: switch sources whenever one of them is slow.
• Every annotated transcript is listed, with the canonical one pre-selected. You choose genomic DNA (with introns) or spliced mRNA, with or without UTRs, and with upstream/downstream flanks of any length.
• Custom sequences: paste any sequence and design on it directly.
• SNP batch import: upload a per-variant flanking-sequence report (.docx, [REF/ALT]-marked) and design flanking primers for every SNP in one go.

Read the sequence

• Sequence map:
  • CDS, UTRs, flanks and introns in their own colours; introns can be collapsed to their length.
  • Hover any base for its position in the gene and its genomic coordinate.
  • Find in sequence: a literal search (optionally the reverse complement too) or an rsID lookup that pins the SNP on the map.
  • Align in sequence: Smith–Waterman finds where a pasted primer or amplicon binds best, mismatches included.
• Feature map: a zoomable overview of exons, introns, CDS and UTRs, with your primers overlaid.
• Exon map: the spliced transcript exon by exon, for placing junction primers where they cross the splice site.

Design primers and probes

By hand. Select bases on any map and right-click:

Pick	What it makes
General	A forward or reverse primer anywhere in the gene
WGA (flank)	Primers in the flanks, to amplify the whole locus
Junction	A primer spanning an exon–exon junction; its partner may sit anywhere in the gene
ARMS twins / common	Allele-specific wild-type/mutant twins with their 3′ end on the SNP, plus the common primer
Probe: general	A TaqMan hydrolysis probe
Probe: allele detection	A wild-type/mutant probe pair differing only at the SNP
BLAST · Secondary structures	For any selection, or any placed primer

Placed primers and probes can be dragged to move or resized by their ends on both the sequence map and the exon map. ARMS twins keep their 3′ end locked on the SNP, allele probes can't lose their SNP, and each mutant partner follows its wild-type twin.

Automatically. Four design modes rank candidate pairs by penalty, each with Tm, GC%, hairpin, self-dimer and heterodimer checks:

Mode	For
General	The best pairs in the gene's exons, introns or anywhere, no other input: matched Tm, least hairpin/dimer structure
WGA	Whole-gene amplification from the flanking regions
Junction	cDNA-specific qRT-PCR primers across exon–exon junctions
SNP/indel	ARMS-PCR allele-specific primer sets

Check every oligo

Strider, a plain-Rust DNA thermodynamics core: nearest-neighbour Tm with salt and Mg²⁺/dNTP corrections, hairpin and dimer ΔG, and ranked suboptimal secondary structures drawn as diagrams. Strider's own picker scans every candidate window, filters on Tm, GC, poly-X runs, hairpins and dimers, and ranks what's left; no Primer3 needed. IDT OligoAnalyzer: one click adds IDT's own Tm next to Strider's, under the same conditions. Your IDT credentials are stored encrypted in your browser (AES-GCM, non-extractable key). NCBI BLAST with identity and query cover for any oligo or region.

One set of reaction conditions is used everywhere: design, analysis, structures and IDT. It matches IDT OligoAnalyzer's qPCR preset:

Na⁺	Mg²⁺	dNTPs	Oligo
50 mM	3 mM	0.8 mM	0.2 µM

Keep track

• My primers gathers every set on the loaded sequence: WGA, general and junction pairs, the ARMS set and the probes. Each shows its amplicon size and Strider numbers, and has Show, Structures, IDT and Remove. Names are editable.
• Primer sets are remembered per sequence. Load the same gene and transcript again and they're back.
• Sessions: save everything to a .primerool.json file, load it later, or pick up an autosave.

Align and design across sequences

• Paste FASTA, GenBank (a whole record or just its numbered sequence lines) or one bare sequence per line. You can also add the loaded sequence, or every other transcript variant of the gene, in one click.
• MAFFT aligns them. The result shows the classic way: a column ruler, one numbered row per sequence, a match line, mismatches highlighted and gaps dimmed.
• Collect homologs across species: give a gene and a taxon (e.g. nirS in Pseudomonas, or a taxonomy ID) and get one coding sequence per species, from GenBank proteins that record where they are coded.
• A conservation track and a list of conserved windows (adjustable identity threshold and length) show where primers can go.
• Design primers in a conserved window or a column range, as individual candidates, as pairs around a target, or as a pair across two windows. Every primer shows how many of the aligned sequences it matches, and how many would fail at its 3′ end.

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Quick start

Use it online

→ primerool.ubch.sci.muni.cz. Nothing to install.

Run it locally

Prerequisites

Tool	Why
Rust (stable)	the server and the thermodynamics engines
A C compiler (tests only)	builds the vendored Primer3 used as a reference in cargo test --workspace
Node.js 20.19+	the frontend (Vite 7)
MAFFT (optional)	multi-sequence alignment

git clone --recurse-submodules https://github.com/KowalskiBio/Primerool.git
cd Primerool

npm run setup   # cargo build (minus the Primer3 reference crates) + frontend npm install
npm run dev     # Rust API on :5050 + Vite on :5173, with hot reload

Then open http://localhost:5173.

Desktop app

The Tauri shell in src-tauri/ runs the same server in-process and opens it in a native window:

cargo install tauri-cli
cargo tauri build     # builds the frontend first, then the native app

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How it works

flowchart LR
    A["🔎 Gene name<br/>accession · sequence"] --> B["📥 Ensembl / NCBI<br/>transcripts, exons, flanks"]
    B --> C["🗺️ Sequence, feature<br/>& exon maps"]
    C --> D["🖱️ Right-click picks<br/>drag to move / resize"]
    C --> E["⚙️ Automatic design<br/>General · WGA · Junction · SNP"]
    D --> F["🌡️ Strider<br/>Tm · GC · hairpins · dimers"]
    E --> F
    F --> G["🧾 My primers<br/>BLAST · structures · IDT"]

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Architecture

The backend is a Rust workspace with one job per crate, and the frontend is a React single-page app. In production, one axum binary serves both the API and the built frontend.

flowchart TB
    UI["React 19 · TypeScript · Tailwind 4<br/><code>frontend/</code>"] -->|JSON over HTTP| S["axum server<br/><code>crates/server</code>"]
    T["Tauri desktop shell<br/><code>src-tauri/</code>"] -.->|runs in-process| S
    S --> E["engine<br/>design · scoring"]
    E --> TC["thermo-core<br/>Strider thermodynamics"]
    S --> PR["providers<br/>Ensembl · NCBI"]
    S --> BL["blast<br/>NCBI BLAST"]
    S --> AL["align<br/>MAFFT"]
    S --> ID["idt<br/>OligoAnalyzer proxy"]
    S --> SN["snp-import<br/>.docx SNP reports"]

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Crate	Role
server	axum HTTP/JSON API: routing, validation and response shaping only
engine	Design modes plus the Strider picker: candidate scan / filter / score / rank
thermo-core	Strider: plain-Rust Tm, salt corrections, hairpin/dimer ΔG, Mathews 2004 folding
primer3-ffi · primer3-sys	Test-only: bindings to the vendored Primer3, used to compare Strider against it (ADR 0001)
providers	SequenceProvider trait with Ensembl and NCBI implementations
blast	NCBI BLAST: submit, poll, fetch, parse
align	MAFFT subprocess wrapper
idt	IDT OligoAnalyzer OAuth2 proxy; credentials are per-request and never stored
snp-import	Parses per-SNP flanking-sequence .docx reports

Repository layout

Primerool/
├── crates/            Rust workspace: server, engine, thermo-core, providers, …
├── frontend/          React + TypeScript + Tailwind single-page app (Vite)
├── src-tauri/         Tauri desktop shell
├── vendor/primer3-py/ Primer3 C sources (git submodule, test reference only)
├── scripts/           setup, dev runner, VM deploy, golden fixtures
├── docs/adr/          architecture decision records
└── docs/images/       README screenshots

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Development

cargo test --workspace                     # Rust unit + parity tests
cargo test -p server --test golden -- --ignored   # replay live API fixtures (network)
npm --prefix frontend run lint             # ESLint
npm --prefix frontend run build            # type-check + production build

The golden fixtures in scripts/golden/fixtures pin the API's responses, so behaviour stays stable across refactors.

Deploying to a server: scripts/deploy_vm.sh fast-forwards the checked-out branch, builds the release server and the frontend, backs up the live install, restarts the systemd service, health-checks it, and rolls back automatically if the check fails.

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Acknowledgements

Primerool stands on the shoulders of MAFFT, the Ensembl REST API, NCBI E-utilities and BLAST, and IDT OligoAnalyzer. Nearest-neighbour parameters follow SantaLucia & Hicks (2004); folding energies follow Mathews et al. (2004). Primer3 (via primer3-py) set the defaults Strider's picker follows and serves as the reference in its tests.

License

Copyright (C) 2026 Vojtech Rejtar.

Primerool is licensed under the GNU Affero General Public License v3.0 or later (AGPL-3.0-or-later). If you modify it and let others use it, including over a network, you must publish your modified source under the same license and keep the original copyright and attribution notices.

Third-party code keeps its own license: crates/thermo-core is derived from Strider (MIT, Copyright (c) 2026 Emilio Venegas), and the vendored Primer3 in vendor/primer3-py is GPL-2.0-or-later.

_{Made for the bench by Vojtěch Rejtar · primerool.ubch.sci.muni.cz}