DNA Melting Temperature Calculator

- May 10, 2025
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Calculate the melting temperature (Tm) of DNA oligonucleotides using various methods.

DNA Sequence

Length: 0 bp
GC Content: 0%

Calculation Method

μM

Display Options

About DNA Melting Temperature

DNA melting temperature (Tm) is the temperature at which 50% of DNA molecules are denatured, with their hydrogen bonds broken, transitioning from double-stranded to single-stranded state.

Calculation Methods
  • Basic (Wallace) Method: A simple rule of thumb for short sequences.
    • Tm = 2°C × (A+T) + 4°C × (G+C)
    Where A, T, G, and C are the counts of the respective nucleotides.
  • Nearest-Neighbor Method: Takes into account interactions between adjacent base pairs.
    • Tm = ΔH / (ΔS + R × ln(c/4)) - 273.15
    Where ΔH is the enthalpy, ΔS is the entropy, R is the gas constant, and c is the primer concentration.
  • Salt-Adjusted Method: Accounts for the effect of salt concentration on stability.
    • Tm = 81.5 + 16.6 × log₁₀[Na⁺] + 0.41 × (%GC) - 600/length
    This is a common formula for longer sequences.
Factors Affecting Melting Temperature
  • GC Content: Higher GC content increases Tm due to the three hydrogen bonds between G-C pairs versus two between A-T pairs
  • Sequence Length: Longer sequences have higher Tm values
  • Salt Concentration: Higher ionic strength stabilises DNA duplex, increasing Tm
  • pH: Acidic or basic conditions can affect the stability of hydrogen bonds
  • Base Mismatches: Non-complementary bases reduce thermal stability
Applications

Understanding DNA melting temperature is critical for many molecular biology techniques:

  • PCR: Designing primers with appropriate Tm values
  • Hybridisation: Setting conditions for DNA or DNA-RNA hybridisation
  • Probe Design: Creating probes for DNA detection assays
  • DNA Sequencing: Optimising conditions for sequencing reactions
  • CRISPR-Cas9: Designing effective guide RNAs
Optimal Annealing Temperature

For PCR and other amplification techniques, the annealing temperature is typically set 5-10°C below the primer's Tm to ensure efficient hybridisation while maintaining specificity.

Understanding the DNA Melting Temperature Calculator

The DNA Melting Temperature Calculator is a handy tool for scientists and students alike. It helps you find out the melting temperature (Tm) of DNA sequences. The melting temperature is vital for many processes in molecular Biology, such as PCR. This calculator uses different methods to give you the most accurate results based on your DNA sequence.

How to Use the Calculator

Using the calculator is straightforward. You just need to enter your DNA sequence in the provided space. The input section is designed for you to easily type or paste your sequence. You can select various calculation methods, which include:

  • Basic (Wallace) Method
  • Nearest-Neighbor Method
  • Salt-Adjusted Method

After entering your details, the calculator will provide you with the melting temperature and Other related information.

What is the Melting Temperature (Tm)?

The melting temperature, or Tm, is the point at which half of the DNA strands are in a double-stranded form, and half are single-stranded. This temperature is crucial for understanding how strong the bonds are between the strands. A higher Tm means stronger binding, which is influenced by factors like GC content and sequence length.

Importance of GC Content

GC content plays a significant role in determining the melting temperature. GC pairs have three hydrogen bonds, while AT pairs only have two. Because of this difference, sequences with higher GC content tend to have higher Tm values. Here are some key points about GC content:

  • Higher GC content increases Tm.
  • Lower GC content decreases Tm.
  • Balancing GC and AT pairs is important for DNA stability.

Selecting the Right Calculation Method

Different methods can be used to calculate Tm based on your needs. The simplest method is the Basic (Wallace) method, which is great for short sequences. However, for longer sequences or specific conditions, consider using the Nearest-Neighbor or Salt-Adjusted methods. Each method takes into account various factors to give you a more accurate melting temperature.

Additional Features of the Calculator

The DNA Melting Temperature Calculator includes several user-friendly features. You can see the length and GC content of your DNA sequence right after inputting it. Moreover, there are options to display helpful information, such as complementary strands and formula details. These features help you understand your results better and improve your experimental design.

Applications of Melting Temperature Calculations

Knowing the melting temperature of DNA is essential for various applications in the lab. Some common uses include:

  • Designing primers for PCR.
  • Setting conditions for DNA hybridization.
  • Creating probes for diagnostics.
  • Optimising sequencing reactions.

Each of these applications benefits from accurate Tm calculations to ensure successful experiments.

Optimal Annealing Temperature for Experiments

When conducting PCR, the annealing temperature is usually set a few degrees below the Tm of the primers. This approach ensures that the primers attach to the DNA targets effectively while avoiding mismatches. Using the calculator helps you set the right conditions for your PCR and other amplification techniques.