Multiplex PCR & Dimer Analysis

Tutorial: Multiplex PCR & Dimer Analysis

Guide for designing multiplex primer sets and analyzing primer dimers.

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Dimer Types

Type	Description	Concern Level
Self-dimer (Homodimer)	Primer binds to itself	Medium
Heterodimer	Two primers bind to each other	High
Hairpin	Primer folds on itself	Low-Medium

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Dimer Matrix Analysis

Check compatibility between multiple primers:

primerlab dimer-matrix --primers primers.json --format text

Input Format

[
  {"name": "GAPDH_F", "sequence": "ATCGATCGATCGATCGATCG"},
  {"name": "GAPDH_R", "sequence": "GCTAGCTAGCTAGCTAGCTA"},
  {"name": "ACTB_F", "sequence": "TGCATGCATGCATGCATGCA"},
  {"name": "ACTB_R", "sequence": "CGATCGATCGATCGATCGAT"}
]

Output Matrix

Dimer Matrix (ΔG kcal/mol)
═══════════════════════════════════════
         GAPDH_F  GAPDH_R  ACTB_F  ACTB_R
GAPDH_F   -2.1     -3.5     -1.2    -0.8
GAPDH_R   -3.5     -1.8     -2.0    -4.1 ⚠️
ACTB_F    -1.2     -2.0     -1.5    -2.3
ACTB_R    -0.8     -4.1 ⚠️  -2.3    -1.9

⚠️ = ΔG < -5.0 (problematic dimer)

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Compatibility Check

Full multiplex compatibility analysis:

primerlab check-compat --primers primers.csv

Features

• Cross-dimer analysis
• Tm compatibility
• Specificity overlap detection
• Multiplex scoring

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Python API

from primerlab.core.compat_check import MultiplexValidator

primers = [
    {"name": "P1", "forward": "ATCG...", "reverse": "GCTA..."},
    {"name": "P2", "forward": "TGCA...", "reverse": "CGAT..."},
]

validator = MultiplexValidator()
result = validator.validate(primers)

print(f"Compatible: {result.is_compatible}")
print(f"Score: {result.compatibility_score}/100")
print(f"Problematic pairs: {result.problematic_dimers}")

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Multiplex Design Tips

Tm Matching

All primers should have similar Tm (within ±2°C):

Good:  P1=60°C, P2=59°C, P3=61°C (Δ2°C)
Bad:   P1=60°C, P2=55°C, P3=65°C (Δ10°C)

Avoid Cross-Reactivity

• Use species-check for each primer pair
• Verify no off-target binding to other amplicons

Amplicon Size Separation

For gel visualization, use distinct sizes:

P1: 150 bp
P2: 250 bp  (+100)
P3: 400 bp  (+150)

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Thresholds

Metric	Good	Warning	Fail
Heterodimer ΔG	> -5.0	-5.0 to -7.0	< -7.0
Homodimer ΔG	> -5.0	-5.0 to -7.0	< -7.0
Hairpin ΔG	> -2.0	-2.0 to -4.0	< -4.0
Tm difference	< 2°C	2-4°C	> 4°C

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See Also

• CLI: check-compat
• CLI: dimer-matrix
• Features: Compatibility Check