Multiplex Primer Comparison
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Thermodynamic Parameters (Nearest Neighbor)
Enter pre-calculated melting temperatures (°C) directly if sequence data is not available.
Multiplex Compatibility Analysis
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°C RECOMMENDED SHARED ANNEALING TEMP (Ta)
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Total Primers
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Lowest Tm
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Highest Tm
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Tm Span
📊 Individual Primer Sequence & Tm Breakdown
Multiplex PCR primer Design Rules
Multiplex PCR allows for simultaneous amplification of multiple loci in a single tube by adding multiple primer pairs. This technique dramatically saves reagents and time, but presents high primer-design complexity.
Key Design Parameters for Multiplexing
- Narrow Tm Span: Melting temperatures of all primers in the mixture must be very close, ideally within 2-3°C of each other, and strictly under 5°C difference.
- Shared Annealing Temperature (Ta): The optimal shared annealing temperature is generally estimated as: Ta = Tm(lowest of all primers) − 5°C. This ensures all primers are capable of stable annealing without causing extreme non-specific reactions for others.
- No Primer-Dimers: Primers must be scanned meticulously for self-complementarity and cross-hybridization to prevent dimer loops that exhaust master mix reagents. Check out our Primer Dimer Checker.
Tm Calculation Scientific Methodology
This calculator employs two standard methods to calculate sequence melting temperatures:
// 1. Wallace Rule (for short oligonucleotides ≤ 13 bp):
Tm = 2 × (A + T) + 4 × (G + C)
// 2. SantaLucia 1998 Nearest-Neighbor Algorithm (for ≥ 14 bp):
Tm = dH / (dS + R × ln(Ct/4)) − 273.15 + 16.6 × log10([Na⁺] / 1000)
where dH and dS are enthalpy/entropy nearest-neighbor parameters, R is the gas constant, and Ct is primer concentration.
Tm = 2 × (A + T) + 4 × (G + C)
// 2. SantaLucia 1998 Nearest-Neighbor Algorithm (for ≥ 14 bp):
Tm = dH / (dS + R × ln(Ct/4)) − 273.15 + 16.6 × log10([Na⁺] / 1000)
where dH and dS are enthalpy/entropy nearest-neighbor parameters, R is the gas constant, and Ct is primer concentration.