Specificity

Specificity Analysis

Ensuring primers amplify only the intended target.

Overview

PrimerLab provides two complementary specificity checks:

Method	Purpose	Speed
In-silico PCR	Validate against template	Fast
BLAST Off-target	Search genome-wide	Slower

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In-silico PCR

Simulates PCR amplification on your template sequence.

What It Checks

1. Primer binding sites — Where primers can anneal
2. Mismatch tolerance — 5’ mismatches allowed, 3’ must match
3. Product prediction — Expected amplicon size and sequence
4. Multiple products — Detects non-specific amplification

Usage

primerlab insilico \
  --primers primers.json \
  --template target.fasta

Binding Rules

Position	Mismatches Allowed
3’ end (last 3 bp)	0
Internal	2
5’ end	2-3

Interpreting Results

✅ 1 product predicted
   720 bp at position 100-820
   Likelihood: 100%

⚠️ 2 products predicted
   Product 1: 720 bp (target)
   Product 2: 450 bp (off-target)

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BLAST Off-target Detection

Searches entire genome for potential binding sites.

What It Checks

1. Sequence similarity — Primers matching other genomic regions
2. Potential amplicons — Off-target primer pairs that could amplify
3. Specificity score — Overall confidence in primer specificity

Usage

primerlab blast \
  --primers primers.json \
  --database genome.fasta

Scoring

Grade	Score	Interpretation
A	90-100	Excellent specificity
B	80-89	Good specificity
C	70-79	Acceptable
D	60-69	Low specificity
F	<60	Poor - redesign recommended

Factors Affecting Score

• Number of off-target hits
• Similarity to off-target regions
• Position of mismatches (3’ is critical)
• Potential for off-target amplification

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Species Specificity

For diagnostic applications, ensure primers don’t cross-react.

Use Cases

• Pathogen detection (detect bacteria, not host)
• GMO testing
• Environmental monitoring

Configuration

specificity:
  target_database: ./pathogen.fasta
  background_database: ./host.fasta
  min_specificity_score: 90

Command

primerlab species-check \
  --primers primers.json \
  --target-db pathogen.fasta \
  --background-db host.fasta

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Best Practices

For High Specificity

1. Check both directions — Forward and reverse independently
2. Use appropriate database — Include all potential cross-reactants
3. Consider 3’ end — Most critical for specificity
4. Test experimentally — In-silico is predictive, not definitive

Database Selection

Application	Database
Human gene amplification	Human genome
Bacterial detection	Host genome + related species
Universal primers	RefSeq microbial

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Workflow Integration

Run specificity checks as part of design:

# Design with automatic validation
primerlab run pcr \
  --config config.yaml \
  --validate \
  --blast --blast-db genome.fasta

Or check existing primers:

# Standalone check
primerlab blast -p primers.json -d genome.fasta
primerlab insilico -p primers.json -t template.fasta

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See Also

• CLI Reference — All specificity commands
• Quality Control — Other QC checks