PCR Primer Design

PCR Primer Design

Comprehensive PCR and qPCR primer design following MIQE 2.0 guidelines with automated validation.

When to Use This Skill

Use this skill when you need:

• ✅ qPCR primers with MIQE 2.0 compliance for publication
• ✅ Standard PCR primers for cloning, genotyping, or amplification (100-1000 bp)
• ✅ TaqMan probes for probe-based qPCR assays
• ✅ Rigorous validation (specificity, dimers, secondary structures)
• ✅ Publication-quality documentation with comprehensive reports

Choose application based on:

• qPCR: 70-140 bp amplicons, strict Tm matching (±2°C), MIQE compliance
• Standard PCR: 100-1000 bp amplicons, general amplification
• TaqMan: Probe-based detection, fluorescent assays
• Multiplex: Multiple targets, compatible Tm requirements
• Sequencing: Single-direction primers, Sanger sequencing

Don't use for:

• ❌ In-situ hybridization probes → use specialized oligo design tools
• ❌ NGS library prep primers → use adapter design workflows
• ❌ CRISPR guide RNAs → use CRISPR-specific design tools

Quick Start (Example)

Test this skill with a sample qPCR design in ~2 minutes:

# Example: Design qPCR primers for a 700bp target sequence
from design_qpcr_primers import design_qpcr_primers

# Sample GAPDH sequence (700 bp, exon 3-4 region)
sequence = "ATGGGGAAGGTGAAGGTCGGAGTCAACGGATTTGGTCGTATTGGGCGCCTGGTCACCAGGGCTGCTTTTAACTCTGGTAAAGTGGATATTGTTGCCATCAATGACCCCTTCATTGACCTCAACTACATGGTTTACATGTTCCAATATGATTCCACCCATGGCAAATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCCCATCACCATCTTCCAGGAGCGAGATCCCTCCAAAATCAAGTGGGGCGATGCTGGCGCTGAGTACGTCGTGGAGTCCACTGGCGTCTTCACCACCATGGAGAAGGCTGGGGCTCATTTGCAGGGGGGAGCCAAAAGGGTCATCATCTCTGCCCCCTCTGCTGATGCCCCCATGTTCGTCATGGGTGTGAACCATGAGAAGTATGACAACAGCCTCAAGATCATCAGCAATGCCTCCTGCACCACCAACTGCTTAGCACCCCTGGCCAAGGTCATCCATGACAACTTTGGTATCGTGGAAGGACTCATGACCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCAAGCTCATTTCCTGGTATGACAACGAATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAGTAAGACCCCTGGACCACCAGCCCCAGCAAGAGCACAAGAGGAAGAGAGAGACCCTCACTGCTGGGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGCCATGTAGACCCCTTGAAGAGGGGAGGGCTCTCTCTTCCTCTTGTGCTCTTGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCAAGCTCATTTCCTGGTATGACAACG"

primers = design_qpcr_primers(
    sequence=sequence,
    amplicon_size_range=(80, 120),
    num_return=5
)

print(f"Found {len(primers['primers'])} primer pairs")
print(f"MIQE-compliant: {sum(p.get('miqe_compliant', False) for p in primers['primers'])}")