Understanding results

Understanding Results

A comprehensive guide to interpreting PrimerLab output, scores, warnings, and recommendations.

Quality Scores (0-100)

Every primer pair receives a quality score based on multiple factors. The score starts at 100 and penalties are applied for sub-optimal characteristics.

Score Categories

Score Range	Grade	Meaning	Action
85-100	✅ Excellent	Optimal design, minimal issues	Use with confidence
70-84	✅ Good	Acceptable, minor issues	Generally safe to use
50-69	⚠️ Fair	Notable issues detected	Review warnings carefully
0-49	❌ Poor	Significant problems	Consider redesign

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Penalty Breakdown

PrimerLab applies penalties for the following issues:

Issue	Penalty (Standard)	What It Means
Primer3 Penalty	-0 to -50	Base penalty from thermodynamic calculations
Hairpin (3’ end)	-15	Primer folds on itself at critical 3’ end
Self-dimer	-15	Primer binds to itself
Hetero-dimer	-10	Forward and reverse primers bind to each other
Weak GC Clamp	-15	No G/C at 3’ end (reduced specificity)
Strong GC Clamp	-5	Too many G/C at 3’ end (may cause mispriming)
Poly-X Run	-10	Repetitive nucleotides (e.g., AAAA, GGGG)

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Understanding Warnings

Warnings do not necessarily mean the primer is unusable. Use this guide to interpret them:

⚠️ Common Warnings

Warning	Severity	Explanation	Recommendation
Hairpin detected	Medium	Secondary structure may form	Check if ΔG > -3 kcal/mol (usually OK)
Self-dimer detected	Medium	Primer may dimerize	Check if ΔG > -6 kcal/mol (usually OK)
Hetero-dimer risk	High	Primers may bind each other	Reduce primer concentration, redesign if ΔG < -8
Weak GC clamp	Low	3’ end lacks G/C	Often acceptable for non-critical PCR
Poly-X run detected	Medium	Repetitive sequence	May cause polymerase slippage, consider masking
Off-target binding	High	BLAST found similar sequences	Verify with in-silico PCR, may need redesign

✅ When Warnings Are Acceptable

1. Hairpin ΔG > -2 kcal/mol: The structure won’t form at PCR temperatures
2. Self-dimer ΔG > -5 kcal/mol: Weak interaction, unlikely to compete with template
3. Weak GC clamp on one primer only: The other primer can compensate
4. Poly-X of 3 or less: Generally not problematic

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Key Metrics Explained

Melting Temperature (Tm)

Tm Difference	Status	Notes
≤ 2°C	✅ Ideal	Both primers anneal at same temperature
2-5°C	⚠️ Acceptable	May need gradient PCR to optimize
> 5°C	❌ Problem	One primer may not anneal efficiently

Recommendation: Keep Tm between 58-62°C for standard PCR, 60-65°C for long-range.

GC Content

GC%	Status	Notes
40-60%	✅ Ideal	Balanced stability
30-40% or 60-70%	⚠️ Acceptable	May need optimization
< 30% or > 70%	❌ Problem	Likely unstable or too tight binding

Delta G (ΔG) Values

ΔG measures the stability of secondary structures. More negative = more stable = more problematic.

ΔG (kcal/mol)	Interpretation
> -2	✅ No significant structure
-2 to -5	⚠️ Minor structure, usually OK
-5 to -8	⚠️ Moderate structure, may affect PCR
< -8	❌ Strong structure, likely to cause problems

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QC Modes

PrimerLab supports three QC stringency levels:

Mode	Use Case	Penalty Severity
strict	Diagnostics, clinical	High penalties, low tolerance
standard	General research	Balanced
relaxed	Exploratory, difficult templates	Lower penalties

Set in config:

qc:
  mode: standard  # or strict, relaxed

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When to Accept vs. Reject

✅ Accept the Primer Pair If

• Score ≥ 70
• No ❌ (critical) warnings
• Tm difference ≤ 3°C
• In-silico PCR produces single product

❌ Reject and Redesign If

• Score < 50
• Multiple high-severity warnings
• Off-target amplification detected
• Tm difference > 5°C
• Product size outside expected range

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See Also

• Scoring System — Detailed algorithm explanation
• Quality Control — QC workflow guide
• Troubleshooting — Common issues and fixes