qPCR Primer and Probe Design - Usage Guide

Overview

This skill co-designs qPCR/RT-qPCR primers and hydrolysis (TaqMan) or molecular-beacon probes with primer3-py. The central idea it enforces: a qPCR assay is a quantitative measurement device, so its validity rests on amplification efficiency (90-110%, standard-curve slope -3.6 to -3.1) and a single product, and every qPCR-specific constraint (short 70-150 bp amplicon, tight Tm, zero dimers, probe Tm offset) exists to protect the efficiency the 2^-ddCq / Pfaffl math assumes. It covers the coupled probe rules (probe Tm 8-10 C above primers, no 5' G, C-rich strand, enforced with PRIMER_INTERNAL_MUST_MATCH_FIVE_PRIME=HNNNN), gDNA exclusion by exon-junction spanning or intron flanking and why processed pseudogenes defeat it, SYBR melt-curve QC, and reference-gene validation. Use it for TaqMan/SYBR assays, exon-spanning expression primers, and multiplex panels; route genome specificity to primer-specificity and dimer checks to primer-validation.

Prerequisites

pip install primer3-py

Input is the target sequence (cDNA for expression assays; mark the splice-junction position if cDNA-specific).
Probe design needs PRIMER_PICK_INTERNAL_OLIGO=1 and raised PRIMER_INTERNAL_* Tm; primer3's internal Tm defaults equal the primer Tm, so they must be set 8-10 C higher.
primer3 has no dedicated no-5'-G option; enforce it with PRIMER_INTERNAL_MUST_MATCH_FIVE_PRIME='HNNNN' (IUPAC H = not G) or a post-hoc filter.
Design-level gDNA exclusion is leaky: processed pseudogenes defeat junction-spanning, so a genome specificity check, DNase, and a no-RT control remain mandatory (primer-specificity).
A passing design is not a validated assay; a standard curve (efficiency, R^2) and controls (NTC, no-RT) are still required (MIQE).

Quick Start

Tell your AI agent what you want to do: - "Design a TaqMan assay for this cDNA: primers around 60 C, probe 8-10 C higher, amplicon under 150 bp, probe not starting with G" - "Design SYBR Green primers for a 100 bp amplicon with minimal dimer potential" - "Make exon-junction-spanning primers so I do not amplify genomic DNA" - "Pick stable reference genes for my qPCR normalization"

Example Prompts

TaqMan assay

"Design a TaqMan assay for my cDNA target: a Tm-matched primer pair near 60 C plus a hydrolysis probe with Tm about 68-70 C, amplicon under 150 bp, and make sure the probe does not start with G and sits on the C-rich strand."

SYBR Green assay

"Design SYBR Green primers for a 90 bp amplicon, tight on 3'-end cross-complementarity to avoid primer-dimers, and remind me to inspect the melt curve for a single peak."

cDNA-specific (avoid gDNA)

"Design exon-junction-spanning primers for this transcript so they will not amplify unspliced genomic DNA, and check whether this gene has a processed pseudogene that would defeat that."

Multiplex and normalization

"Design qPCR assays for my target and two reference genes with matched Tm and efficiency, screen all primers and probes for cross-dimers, and tell me how to validate the reference genes with geNorm."

What the Agent Will Do

Load the target sequence and confirm the assay type (TaqMan vs SYBR, expression vs genomic).
Co-design primers and (for TaqMan) an internal probe: short amplicon, Tm-matched primers, probe Tm raised 8-10 C, no 5'-G via HNNNN.
For expression assays, add exon-junction spanning and flag the processed-pseudogene caveat.
Report the primer/probe set with Tm offsets and amplicon size, and note that SYBR needs a melt-curve check.
Route the chosen pair to primer-specificity (genome/pseudogene) and primer-validation (dimers), and state the standard-curve/efficiency and control requirements (MIQE).
For multiplex, check all primer/probe pairs for cross-dimers and recommend matched efficiencies and primer-limiting.

Tips

Keep the amplicon 70-150 bp; long amplicons lose efficiency and bend the standard curve.
Raise the probe Tm 8-10 C above the primers so it is bound when the polymerase cleaves it; the internal defaults equal the primer Tm and must be changed.
Forbid a 5'-G on the probe and prefer the C-rich strand; a 5'-G quenches the reporter.
Do not trust exon-junction spanning alone for gDNA exclusion; check the genome for pseudogenes and keep DNase plus a no-RT control.
For SYBR, the melt curve is the specificity readout; a low-Tm shoulder means primer-dimer.
Validate reference genes with geNorm/NormFinder in the actual experimental conditions; never normalize to a single unvalidated gene.
Efficiency is part of the quantification equation; run a standard curve and use Pfaffl if target and reference efficiencies are not matched.

primer-basics - Design fundamentals, Tm matching, and the constraint model
primer-validation - Dimers/hairpins of primers and probe at reaction conditions
primer-specificity - Genome/pseudogene specificity and gDNA exclusion checking
sequence-manipulation/transcription-translation - Work with cDNA and reading frames
differential-expression/deseq2-basics - Downstream analysis qPCR validates against