Tm Calculator (Modified Allawi & SantaLucia Method)

Thermo Fisher Online Tm Calculator

Modified Allawi & SantaLucia Method:

\[ Tm = \frac{\Delta H}{\Delta S + R \times \ln(\frac{C_T}{4})} - 273.15 + 16.6 \times \log_{10}([Na^+]) \]

Melting Temperature (Tm):

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1. What is the Modified Allawi & SantaLucia Method?

The modified Allawi & SantaLucia method is a thermodynamic approach for calculating oligonucleotide melting temperatures (Tm) that considers nearest-neighbor interactions, salt concentrations, and oligonucleotide concentration. It provides more accurate Tm predictions than simpler methods.

2. How Does the Calculator Work?

The calculator uses the following equation:

\[ Tm = \frac{\Delta H}{\Delta S + R \times \ln(\frac{C_T}{4})} - 273.15 + 16.6 \times \log_{10}([Na^+]) \]

Where:

\( \Delta H \) — Enthalpy change (kcal/mol)
\( \Delta S \) — Entropy change (cal/mol·K)
\( R \) — Gas constant (1.9872 cal/mol·K)
\( C_T \) — Total oligonucleotide concentration (M)
\( [Na^+] \) — Sodium ion concentration (M)

Explanation: The method calculates thermodynamic parameters based on nearest-neighbor interactions between base pairs, then applies corrections for concentration and salt effects.

3. Importance of Tm Calculation

Details: Accurate Tm prediction is crucial for PCR primer design, hybridization experiments, and other molecular biology applications where precise temperature control is needed.

4. Using the Calculator

Tips: Enter the DNA sequence (ACGT only), oligonucleotide concentration (typically 0.1-1.0 μM), and sodium concentration (typically 50 mM for standard PCR). The sequence should be 15-30 bases for optimal results.

5. Frequently Asked Questions (FAQ)

Q1: Why use this method instead of the Wallace rule?
A: This method considers sequence-specific thermodynamics and salt effects, providing more accurate predictions than simple GC-content based rules.

Q2: What are typical Tm values for PCR primers?
A: Optimal primer Tms are typically 55-65°C, with primer pairs within 2-3°C of each other.

Q3: How does salt concentration affect Tm?
A: Higher salt concentrations stabilize DNA duplexes, increasing Tm by about 16.6°C per 10-fold increase in [Na+].

Q4: Are there limitations to this method?
A: Accuracy decreases for very short (<15 bases) or long (>30 bases) oligonucleotides, and for sequences with unusual secondary structure.

Q5: Should I use this for RNA sequences?
A: No, this calculator is for DNA only. RNA duplexes have different thermodynamic parameters.