SYBR Green Melt Curve Analysis

Feature documentation: SYBR Green Melt Curve Analysis

Predict melt curve characteristics for SYBR Green qPCR amplicons.

Overview

The melt curve module predicts:

• Amplicon Tm - Melting temperature of the PCR product
• Melt Profile - -dF/dT curve shape
• Peak Detection - Single vs multiple peaks
• Quality Score - Assessment of amplicon suitability

Why Melt Curve Analysis?

SYBR Green binds to all double-stranded DNA. Melt curve analysis helps:

1. Confirm Specificity - Single peak = single product
2. Detect Primer-Dimers - Lower Tm peaks
3. Identify Non-Specific - Multiple peaks

Python API

predict_melt_curve

from primerlab.core.qpcr import predict_melt_curve

result = predict_melt_curve(
    amplicon_sequence="ATGC" * 25,  # 100bp
    na_concentration=50.0,
)

print(f"Predicted Tm: {result.predicted_tm}°C")
print(f"Single peak: {result.is_single_peak}")
print(f"Grade: {result.grade}")

Report Generation

from primerlab.core.qpcr import (
    predict_melt_curve,
    generate_melt_markdown,
    generate_melt_csv,
)

result = predict_melt_curve("ATGC" * 25)

# Markdown report
md = generate_melt_markdown(result)

# CSV data for plotting
csv = generate_melt_csv(result)

Optimal Amplicon Properties

Property	Optimal	Acceptable
Length	80-120 bp	70-200 bp
GC%	40-60%	30-70%
Tm	80-88°C	75-92°C

Interpreting Results

Single Peak (Grade A-B)

✅ Indicates specific amplification:

• One clean melt peak
• Sharp transition
• Consistent Tm between replicates

Multiple Peaks (Review Required)

⚠️ May indicate:

• Lower peak - Primer-dimers
• Higher peak - Wrong target
• Shoulder - Non-specific products

Output

MeltCurveResult

@dataclass
class MeltCurveResult:
    amplicon_sequence: str
    predicted_tm: float
    tm_range: Tuple[float, float]
    peaks: List[MeltPeak]
    melt_curve: List[Dict]  # {temperature, derivative}
    is_single_peak: bool
    quality_score: float
    grade: str
    warnings: List[str]

MeltPeak

@dataclass
class MeltPeak:
    temperature: float
    height: float      # Relative (0-1)
    width: float       # °C at half-max
    is_primary: bool

Grading

Grade	Score	Interpretation
A	≥90	Excellent - clean single peak
B	80-89	Good - single peak with minor issues
C	70-79	Acceptable - review recommended
D	60-69	Poor - may have non-specific products
F	<60	Fail - redesign primers

See Also

• Probe Binding
• qPCR Amplicon QC