Pcr design

PCR Primer Design Guide

A complete walkthrough of the PCR primer design workflow.

Overview

This guide covers:

1. Configuration setup
2. Running the design
3. Understanding output
4. Validating primers
5. Exporting for ordering

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1. Prepare Your Sequence

PrimerLab accepts sequences in two ways:

Option A: Inline sequence

input:
  sequence: "ATGCGATCGATCG..."

Option B: FASTA file

input:
  sequence_path: /path/to/gene.fasta

---

2. Configure Parameters

Create pcr_config.yaml:

input:
  sequence_path: ./my_gene.fasta

parameters:
  # Melting temperature
  tm:
    min: 57.0
    opt: 60.0
    max: 63.0
  
  # Primer length
  primer_size:
    min: 18
    opt: 20
    max: 25
  
  # Amplicon size
  product_size:
    min: 200
    max: 500
  
  # GC content
  gc:
    min: 40.0
    max: 60.0

output:
  format: json
  directory: ./output

Parameter Recommendations

Application	Product Size	Tm	Notes
Cloning	200-2000 bp	58-62°C	Standard
Colony PCR	300-1000 bp	55-60°C	Fast cycling
Long-range	2000-10000 bp	62-68°C	Long primers
Diagnostic	100-500 bp	58-62°C	Specific

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3. Run the Design

primerlab run pcr --config pcr_config.yaml

Available Options

primerlab run pcr \
  --config pcr_config.yaml \
  --out results/ \
  --export idt,benchling \
  --report \
  --report-format html

Flag	Purpose
--out	Custom output directory
--export	Generate ordering files
--report	Generate enhanced report
--mask auto	Exclude repeat regions
--validate	Run in-silico PCR
--blast	Check off-targets

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4. Understand the Output

Output Files

File	Description
primers.json	Full results with all metrics
report.md	Human-readable summary
primers_idt.csv	IDT ordering format
audit.json	Design parameters used

Primer Quality Scores

Each primer pair receives a score (0-100) based on:

Factor	Weight	Criteria
Tm match	25%	Closeness to optimal
GC content	20%	Within 40-60%
Self-dimer	20%	ΔG > -5 kcal/mol
Hairpin	15%	ΔG > -2 kcal/mol
3’ stability	20%	No runs of G/C

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5. Validate Primers

In-silico PCR

Verify primers amplify the correct region:

primerlab insilico \
  -p output/primers.json \
  -t my_gene.fasta

Off-target Check

Check specificity against a genome:

primerlab blast \
  -p output/primers.json \
  -d /path/to/genome.fasta

Compatibility Check

Verify no primer dimers:

primerlab check-compat -p output/primers.json

---

6. Export for Ordering

Generate files for synthesis vendors:

# IDT format
primerlab run pcr -c config.yaml --export idt

# Multiple formats
primerlab run pcr -c config.yaml --export idt,sigma,thermo

Supported Vendors

Vendor	Format	Notes
IDT	CSV	Includes scale/purification
Sigma	CSV	Ready for upload
Thermo	XLSX	Excel format
Benchling	CSV	API-compatible

---

Complete Example

# 1. Create config
cat > gene_amplification.yaml << 'EOF'
input:
  sequence_path: ./BRCA1_exon1.fasta

parameters:
  tm:
    opt: 60.0
  product_size:
    min: 200
    max: 400
  gc:
    min: 45.0
    max: 55.0

output:
  include_rationale: true
EOF

# 2. Design primers
primerlab run pcr \
  --config gene_amplification.yaml \
  --validate \
  --report \
  --export idt

# 3. Review results
cat primerlab_output/report.md

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Troubleshooting

No primers found

• Relax constraints (wider Tm range, larger product size)
• Check sequence for repeat regions (primerlab stats gene.fasta)

Poor quality scores

• Sequence may have challenging regions (high GC, repeats)
• Try --mask auto to avoid problematic areas

Primers form dimers

• Run primerlab check-compat on results
• Choose pairs with higher compatibility scores

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Next Steps

• qPCR Design — Add TaqMan probes
• Batch Processing — Design for multiple genes
• Nested PCR — Increased specificity
• Multiplex PCR — Multiple targets
• Species Specificity — Pathogen detection