DNA Melting Temperature Calculator

Category: Biology
- May 22, 2025
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DNA Melting Temperature Calculator

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 stabilizes 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
  • Hybridization: Setting conditions for DNA or DNA-RNA hybridization
  • Probe Design: Creating probes for DNA detection assays
  • DNA Sequencing: Optimizing 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 hybridization while maintaining specificity.

Understanding DNA Melting Temperature

DNA melting temperature, or Tm, is a key concept in molecular Biology. It’s the temperature at which half of the DNA molecules separate into single strands. This is important for many lab techniques, as it helps scientists know how to handle DNA samples. Knowing the Tm can make processes like PCR and hybridization more effective.

Getting Started with the DNA Melting Temperature Calculator

This DNA Melting Temperature Calculator makes it easy to find the Tm of your DNA sequences. Simply enter your DNA sequence, and choose a calculation method. You can select between basic, nearest-neighbour, or salt-adjusted methods, depending on the specifics of your experiment. This flexibility helps ensure accurate results.

Why Choose Different Methods?

There are several methods for calculating melting temperature, and they each have their uses:

  • Basic (Wallace) Method: A simple calculation for short sequences based on the number of A, T, G, and C bases.
  • Nearest-Neighbor Method: This considers how the bases interact with each Other, providing a more accurate estimate for longer strands.
  • Salt-Adjusted Method: This takes into account the concentration of salt, which can affect the stability of the DNA double helix.

Inputting Your DNA Sequence

When using the calculator, you'll need to enter your DNA sequence from 5' to 3'. Be sure to check the length and GC content, as they both impact the Tm. The calculator will provide real-time updates, showing your sequence's length and GC content as you type. This ensures you have all the information you need for accurate results.

Understanding Results

Once you enter your data, the calculator will display the melting temperature and optimal annealing temperature. This is crucial for experiments that require precise temperature settings. Additionally, the calculator can show complementary strands, helping you visualize and understand your sequences better.

Factors Influencing Melting Temperature

Several factors can affect the melting temperature of DNA:

  • GC Content: Higher GC content increases Tm because G-C pairs form three hydrogen bonds compared to A-T pairs, which only have two.
  • Sequence Length: Longer DNA sequences tend to have a higher melting temperature.
  • Salt Concentration: More salt stabilizes the DNA strands, leading to a higher Tm.

Real-World Applications of Tm

Understanding melting temperature is essential for various applications in molecular biology. Some of these include:

  • Designing primers for PCR.
  • Setting conditions for hybridization tests.
  • Creating probes for detecting specific DNA sequences.
  • Optimizing conditions for DNA sequencing.
  • Designing guide RNAs for CRISPR-Cas9 Technology.

Optimising Your Experimental Conditions

For successful experiments, it's best to set the annealing temperature around 5-10°C below the Tm of the primers. This ensures effective binding while reducing non-specific interactions. Using the DNA Melting Temperature Calculator will help you find the right Tm, making your experiment more reliable.