1. What is the Tm - modified Allawi & SantaLucia method?

The modified Allawi & SantaLucia method calculates DNA melting temperature (Tm) based on nearest-neighbor thermodynamics. It provides more accurate results than simple Wallace rules by considering sequence-specific parameters and salt corrections.

2. How Does the Calculator Work?

The calculator uses the modified Allawi & SantaLucia equation:

Where:

• \( \Delta H \) — Enthalpy change (kcal/mol)
• \( \Delta S \) — Entropy change (cal/mol·K)
• \( R \) — Gas constant (1.987 cal/mol·K)
• \( C_T \) — Total oligo concentration (M)
• \( [salt] \) — Salt concentration (M)

Explanation: The equation accounts for sequence-specific thermodynamics and salt effects on DNA melting behavior.

3. Importance of Tm Calculation

Details: Accurate Tm prediction is crucial for PCR primer design, hybridization experiments, and molecular biology applications requiring specific annealing temperatures.

4. Using the Calculator

Tips: Enter DNA sequence (5' to 3'), salt concentration (typically 0.05M for standard PCR), and oligo concentration (typically 250nM for PCR). Sequence should contain only A, T, C, G characters.

5. Frequently Asked Questions (FAQ)

Q1: Why use this method instead of basic %GC calculation?
A: This method provides more accurate results by considering sequence context and nearest-neighbor interactions, not just overall GC content.

Q2: What are typical Tm values for PCR primers?
A: Optimal primer Tms are typically 55-65°C, with forward and reverse primers within 2°C of each other.

Q3: How does salt concentration affect Tm?
A: Higher salt concentrations stabilize DNA duplexes, increasing Tm. The effect is logarithmic.

Q4: What concentration units should I use?
A: Use molar (M) units for both salt and oligo concentration (e.g., 0.05M salt = 50mM).

Q5: Does this work for RNA/DNA hybrids?
A: No, this calculator is for DNA/DNA duplexes. RNA/DNA hybrids require different parameters.