/primer-design

name: primer-design
description: Designs and audits PCR and RT-qPCR primers with Primer3, explicit thermodynamic conditions, reference-based off-target amplification searches, and traceable sequence coordinates. Use for designing primer pairs, checking existing primers, exon-junction or isoform-specific assays, variant masking, cloning tails, multiplex compatibility, and interpreting Primer-BLAST results. Includes bounded local in-silico PCR and BLAST screening; distinguishes computational candidates from experimentally validated assays.
license: MIT
compatibility: Requires Python 3.11+ and primer3-py 2.3.1 for design and thermodynamics. Local exhaustive screening uses the standard library; BLAST screening additionally needs blastn and makeblastdb on PATH. Network access is needed only for installation, reference retrieval, or external Primer-BLAST.
metadata:
  version: "1.1"
  skill-author: K-Dense Inc.
  last-reviewed: "2026-10-01"

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

| Request | Start here | | --- | --- | | New genomic PCR or RT-qPCR pair | Define the assay and reference; design; assess thermodynamics; screen products. | | Check an existing pair | Prepare pair TSV; assess both full oligos and annealing cores; screen with explicit intended coordinates. | | Exon junction, transcript isoform, allele discrimination | Read [design-workflows.md](references/design-workflows.md); supply sequence annotation before imposing constraints. | | Cloning/adaptor-tailed primers | Design annealing cores, append declared 5-prime tails, reassess full oligos, reconstruct the final product. | | Multiplex panel | Enable `--multiplex` in both thermodynamics and specificity tools to assess oligo interactions and cross-pair products. | | Degenerate, bisulfite, probe, or modified-base assay | Use the specialized workflow in [design-workflows.md](references/design-workflows.md); the bundled ordinary-DNA model is insufficient. |

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](references/input-contract.md) for file schemas and coordinate examples. Copy [assay-report-template.md](assets/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.

Read [sources.md](references/sources.md) when updating dependencies or API assumptions. The scripts record engine versions and effective settings in their JSON reports.

1. Design candidates

For an initial functional demonstration, use the bundled synthetic sequence. It is nonbiological example input, not an experimentally validated assay:

.venv-primer/bin/python "$SKILL_DIR/scripts/design_primers.py" \
  --template "$SKILL_DIR/assets/demo-template.fasta" \
  --preset qpcr --config "$SKILL_DIR/assets/qpcr-config.json" \
  --output design.json --pai