Primer Validation - Usage Guide

Overview

This skill validates chosen PCR/qPCR oligos for intramolecular thermodynamic liabilities with primer3-py: hairpins, self-dimers, cross-dimers, and 3'-end stability. The central idea it enforces: a "dimer-free" verdict is a thermodynamic prediction at the supplied salt, Mg2+, dNTP, and oligo conditions and the temperature of evaluation, and the 3' end is the lethal locus because a 3'-anchored dimer is polymerase-extendable into primer-dimer. So structures are ranked by their dG at the annealing temperature and by 3'-end involvement, not by a single global score. Use it to check primer pairs before ordering, troubleshoot dimers or smears, or screen many oligos; route genome-wide off-target checking to primer-specificity and primer design to primer-basics.

Prerequisites

pip install primer3-py

Input is one or more oligo sequences (primers and/or a probe), as strings.
Supply the real reaction conditions: monovalent salt, Mg2+, dNTP, and oligo concentration, plus temp_c set to the annealing temperature. The same primer reads "fine" or "dimer-prone" depending on these.
ThermoResult dG is in cal/mol (divide by 1000 for kcal/mol), and .structure_found must be checked before trusting any number.
This skill checks the oligos against themselves and each other; it does NOT check whether they bind off-target in the genome (that is primer-specificity).

Quick Start

Tell your AI agent what you want to do: - "Check this primer pair for hairpins and dimers at my qPCR conditions (50 mM monovalent, 3 mM Mg, anneal 60 C)" - "Is the 3' end of my forward and reverse primer forming a stable dimer?" - "Screen these 12 oligos and flag any with strong secondary structure" - "My PCR gives a low-molecular-weight band; could it be a primer-dimer?"

Example Prompts

Pre-order pair check

"Validate this forward/reverse pair before I order them. Use my reaction conditions (50 mM K, 3 mM Mg, 0.8 mM dNTP, 250 nM primer) and evaluate at the 60 C anneal step. Tell me the hairpin, homodimer, and heterodimer dG and whether the 3' ends pair."

Troubleshoot a primer-dimer

"I see a strong low-Tm peak in my SYBR melt curve and a small band on the gel. Check whether my primers form a 3'-end heterodimer that could be extending into primer-dimer, and show the structure."

Batch screening

"Here are 20 candidate primers. Compute hairpin and homodimer Tm for each with the fast Tm-only functions and give me the ones above 45 C to inspect in detail."

Tailed primers

"My primers carry EcoRI and BamHI 5' tails. Validate the full tailed oligos for dimers, since the restriction sites are palindromic."

What the Agent Will Do

Read the oligo sequence(s) and the reaction conditions (salt, Mg2+, dNTP, oligo concentration, annealing temperature).
Run calc_hairpin and calc_homodimer on each oligo and calc_heterodimer on the pair at those conditions and temp_c.
Compute calc_end_stability to expose 3'-end-anchored, extendable dimers and print the ASCII structure so the 3'-end pairing is visible.
Gate every result on .structure_found and convert dG from cal/mol to kcal/mol before comparing to thresholds.
Compare the two primer Tms and flag a mismatch greater than ~2 C.
Report flags as conditions-dependent (not absolute pass/fail) and route a redesign to primer-basics or an off-target check to primer-specificity.

Tips

Evaluate at the annealing temperature (set temp_c), not the 37 C default; a hairpin that melts well below Ta is harmless.
Weight 3'-end structures most heavily; a weak dimer that pairs the 3' ends beats a strong one with free 3' ends for causing artifact.
Convert dG to kcal/mol (divide cal/mol by 1000) before applying any threshold.
Check the heterodimer explicitly; two individually clean primers can still cross-dimer.
For a tailed primer, validate the full oligo, not just the binding core; palindromic sites and Gibson arms dimerize.
Treat thresholds as flags for inspection, not verdicts; read the ASCII structure and judge at the real conditions.

primer-basics - Design Tm-matched primer pairs (redesign if validation fails)
primer-specificity - Genome-wide off-target / in-silico PCR (a different question)
qpcr-primers - Co-design qPCR primers and probes, including probe self-structure
sequence-manipulation/seq-objects - Reverse-complement and assemble tailed oligos to validate