Primer design and specificity

Produce candidate oligos in 5-prime-to-3-prime orientation, with the exact template, chemistry, intended products, and search scope behind each conclusion. Calculate sequence-dependent quantities with the supplied tools. A familiar gene name, good Primer3 penalty, or a single BLAST alignment cannot establish primer specificity.

Choose the workflow

The local tooling supports paired primers with unambiguous ACGT cores. Advanced assay types have substantive design and validation guidance, but are not silently reduced to ordinary PCR. This skill designs assays; expression normalization, experimental diagnostic validation, and guide-RNA design are separate tasks.

Establish the assay contract

Obtain what changes the result; use explicit provisional assumptions for an exploratory design, and identify them in the report:

• Purpose and template: genomic DNA, cDNA, plasmid, or another defined substrate; target organism, accession with version, assembly/transcript release, strand, desired isoforms, and product-size range. Name wanted and unwanted templates.
• Sequence evidence: local FASTA plus source/retrieval date and its SHA-256 hash. A locus excerpt uses local coordinates; record its mapping to the full reference. Include relevant paralogs, pseudogenes, alternate contigs, transcript isoforms, vector backbone, and host sequence in the appropriate screen.
• Reaction conditions: polymerase/buffer, monovalent salt, total divalent salt, total dNTP, and oligo concentrations. Primer3 uses mM for salts/dNTP and nM for DNA. Record the initial reaction concentration separately from Primer3's effective annealing-oligo concentration parameter. Engine defaults are assumptions.
• Constraints: target interval, allowed/excluded binding regions, junctions, variant exclusions and their source, fixed primers, tails, or multiplex membership. Do not guess exon boundaries or silently substitute another assembly.
• Definition of an acceptable result: relevant off-target references, amplicon lengths, mismatch search limits, controls, and experimental validation appropriate to the assay. There is no universal thermodynamic cutoff that validates all PCRs.

Use input-contract.md for file schemas and coordinate examples. Copy assay-report-template.md into the analysis directory to collect evidence. Reference retrieval may be manual or through an established sequence API; preserve accession/version and verify the returned sequence. The supplied scripts use local files and do not submit sequences online.

Install and verify

Set SKILL_DIR to this skill's actual installed directory. Work in a separate analysis directory so environments, reference databases, and results do not enter the skill.

uv venv --python 3.13 .venv-primer
uv pip install --python .venv-primer/bin/python -r "$SKILL_DIR/assets/requirements.txt"
.venv-primer/bin/python "$SKILL_DIR/scripts/design_primers.py" --help

Use the environment's Scripts/python.exe on Windows. The design/thermodynamic examples target primer3-py 2.3.1, tested with Python 3.13. For the optional BLAST engine, install NCBI BLAST+ from its official distribution and check:

blastn -version
makeblastdb -version

Local integration checks also exercise BLAST+ 2.17.0; other releases require checking their output and search behavior before claiming equivalent coverage.

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