DNA Melting Temperature Calculator
The melting temperature (Tm) of a DNA oligonucleotide is the temperature at which half of the DNA strands are denatured (separated into single strands) and half remain in their double-stranded form. This critical parameter is essential in molecular biology applications such as Polymerase Chain Reaction (PCR), hybridization assays, and primer design.
Understanding and accurately predicting Tm helps ensure the specificity and efficiency of these experiments. Factors influencing Tm include the length of the oligonucleotide, its GC content (the percentage of Guanine and Cytosine bases), and the concentration of monovalent cations like sodium ions (Na+) in the solution.
Guanine (G) and Cytosine (C) bases form three hydrogen bonds, making their pairing stronger than Adenine (A) and Thymine (T) which form two hydrogen bonds. Therefore, a higher GC content leads to a higher melting temperature. Similarly, longer oligonucleotides require more energy to separate, resulting in a higher Tm. Monovalent cations stabilize the DNA double helix by shielding the negatively charged phosphate backbone, thus increasing the Tm.
This calculator uses a commonly accepted formula for oligonucleotides longer than 13 base pairs, which takes into account the GC content, oligonucleotide length, and sodium ion concentration to estimate the melting temperature.
How to Use This Calculator:
- Guanine (G) Count: Enter the total number of Guanine bases in your DNA oligonucleotide.
- Cytosine (C) Count: Enter the total number of Cytosine bases in your DNA oligonucleotide.
- Adenine (A) Count: Enter the total number of Adenine bases in your DNA oligonucleotide.
- Thymine (T) Count: Enter the total number of Thymine bases in your DNA oligonucleotide.
- Sodium Ion Concentration (mM): Input the concentration of sodium ions (Na+) in your solution, typically in millimolar (mM). Common PCR buffer concentrations are around 50 mM.
- Click "Calculate Melting Temperature" to see the estimated Tm.
Example Calculation:
Let's consider a DNA oligonucleotide with the following characteristics:
- Guanine (G) Count: 10
- Cytosine (C) Count: 10
- Adenine (A) Count: 5
- Thymine (T) Count: 5
- Sodium Ion Concentration: 50 mM
Step 1: Calculate Total Length (N)
N = G + C + A + T = 10 + 10 + 5 + 5 = 30 base pairs
Step 2: Calculate GC Content (%)
%GC = ((G + C) / N) * 100 = ((10 + 10) / 30) * 100 = (20 / 30) * 100 ≈ 66.67%
Step 3: Convert Sodium Ion Concentration to Molar (M)
[Na+]_M = 50 mM / 1000 = 0.05 M
Step 4: Apply the Melting Temperature Formula
Using the formula: Tm = 64.9 + 0.41 * (%GC) – (675 / N) + 16.6 * log10([Na+]_M)
Tm = 64.9 + 0.41 * 66.67 – (675 / 30) + 16.6 * log10(0.05)
Tm = 64.9 + 27.3347 – 22.5 + 16.6 * (-1.301)
Tm = 64.9 + 27.3347 – 22.5 – 21.5966
Tm ≈ 48.14 °C
Thus, the estimated melting temperature for this oligonucleotide is approximately 48.14 °C.
function calculateMeltingTemperature() {
var guanineCount = parseFloat(document.getElementById("guanineCount").value);
var cytosineCount = parseFloat(document.getElementById("cytosineCount").value);
var adenineCount = parseFloat(document.getElementById("adenineCount").value);
var thymineCount = parseFloat(document.getElementById("thymineCount").value);
var sodiumConcentration_mM = parseFloat(document.getElementById("sodiumConcentration").value);
var resultDiv = document.getElementById("result");
resultDiv.innerHTML = ""; // Clear previous results
// Input validation
if (isNaN(guanineCount) || isNaN(cytosineCount) || isNaN(adenineCount) || isNaN(thymineCount) || isNaN(sodiumConcentration_mM)) {
resultDiv.innerHTML = "Please enter valid numbers for all fields.";
return;
}
if (guanineCount < 0 || cytosineCount < 0 || adenineCount < 0 || thymineCount < 0) {
resultDiv.innerHTML = "Base counts cannot be negative.";
return;
}
if (sodiumConcentration_mM 13 bp
// Tm = 64.9 + 0.41 * (%GC) – 675 / N + 16.6 * log10([Na+])
var tm = 64.9 + (0.41 * gcContent) – (675 / totalLength) + (16.6 * Math.log10(sodiumConcentration_M));
resultDiv.innerHTML = "Estimated Melting Temperature (Tm):
" + tm.toFixed(2) + " °C";
}
// Add Math.log10 polyfill for older browsers if necessary
if (!Math.log10) {
Math.log10 = function(x) {
return Math.log(x) / Math.LN10;
};
}
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