Tm Calculator

DNA/RNA Oligonucleotide Melting Temperature (Tm) Calculator

Please enter an oligonucleotide sequence.

Please enter a valid positive monovalent cation concentration.

Warning: Both T and U found. Assuming T is replaced by U for calculation.

The sequence contains no valid DNA/RNA bases (A, T, G, C, U).

" + tm.toFixed(2) + " °C

Sequence Length (N): " + N + "

GC Content: " + percentGC.toFixed(1) + "%

Monovalent Cation: " + monovalentCation + " mM

Note: This formula may be less accurate for very short oligonucleotides (<14 bp).

Understanding DNA/RNA Oligonucleotide Melting Temperature (Tm)

The Melting Temperature (Tm) of a DNA or RNA oligonucleotide is a critical parameter in molecular biology, representing the temperature at which half of the DNA (or RNA) strands are denatured (separated into single strands) and half remain in their double-stranded form. This value is crucial for designing experiments involving nucleic acid hybridization, such as Polymerase Chain Reaction (PCR), quantitative PCR (qPCR), DNA sequencing, microarrays, and probe-based assays.

Why is Tm Important?

• PCR Primer Design: For successful PCR, primers must bind specifically to their target DNA sequence. The annealing temperature during PCR is typically set a few degrees below the Tm of the primers. If the annealing temperature is too high, primers won't bind; if too low, they might bind non-specifically, leading to unwanted products.
• Probe Hybridization: In techniques like Southern blotting, Northern blotting, or fluorescence in situ hybridization (FISH), probes need to hybridize efficiently to their target sequences. Knowing the Tm helps optimize hybridization conditions for specificity and sensitivity.
• Oligonucleotide Stability: Tm provides an indication of the stability of a DNA/RNA duplex. Higher Tm values generally mean a more stable duplex.

Factors Affecting Tm

Several factors influence the melting temperature of an oligonucleotide:

1. Oligonucleotide Length (N): Longer oligonucleotides generally have higher Tm values because more hydrogen bonds need to be broken to separate the strands.
2. GC Content (%GC): Guanine (G) and Cytosine (C) bases form three hydrogen bonds, while Adenine (A) and Thymine (T) (or Uracil (U) in RNA) form two. Therefore, sequences with a higher percentage of G-C pairs are more stable and have a higher Tm.
3. Monovalent Cation Concentration (e.g., Na+, K+): Cations (like Na+ or K+) help neutralize the negative charges on the phosphate backbone of DNA, reducing electrostatic repulsion between the strands. Higher salt concentrations stabilize the duplex, leading to a higher Tm.
4. Formamide or DMSO Concentration: These denaturing agents destabilize DNA duplexes, lowering the Tm. They are often used to reduce non-specific binding. (This calculator does not account for these agents).
5. Oligonucleotide Concentration: While less significant than other factors for typical primer concentrations, very high oligonucleotide concentrations can slightly increase Tm due to increased chances of re-annealing. (This calculator uses a common formula that does not explicitly include oligo concentration, assuming typical experimental ranges).

How This Calculator Works

This calculator uses a widely accepted empirical formula to estimate the Tm for DNA/RNA oligonucleotides, particularly suitable for primers and probes in the range of 14-200 base pairs. The formula takes into account the oligonucleotide's length, its GC content, and the monovalent cation concentration:

Tm = 81.5 + 0.41 * (%GC) - (675 / N) + 16.6 * log10([Na+])

• %GC: The percentage of Guanine and Cytosine bases in the sequence (0-100).
• N: The total length of the oligonucleotide in base pairs.
• [Na+]: The monovalent cation concentration in Molar (e.g., 50 mM = 0.05 M).

Please note that Tm calculations are estimations, and different formulas may yield slightly different results. This formula provides a good practical estimate for many molecular biology applications.

How to Use the Calculator

1. Oligonucleotide Sequence: Enter your DNA or RNA sequence (e.g., ATGCGTACGT). The calculator will automatically ignore any non-standard characters and convert 'U' to 'T' for calculation if 'U' is present, or treat 'T' as 'T' if 'U' is absent.
2. Monovalent Cation Concentration (mM): Input the concentration of monovalent cations (like Na+ or K+) in your reaction buffer, typically in millimolar (mM). Common PCR buffers might have 50-100 mM monovalent cations.
3. Calculate Tm: Click the "Calculate Tm" button to get the estimated melting temperature in degrees Celsius (°C).

Example Calculation

Let's calculate the Tm for the sequence AGCTAGCTAGCTAGCT with a monovalent cation concentration of 50 mM:

• Sequence: AGCTAGCTAGCTAGCT
• Length (N): 16 base pairs
• GC Count: 8 (G=4, C=4)
• %GC: (8 / 16) * 100 = 50%
• Monovalent Cation Concentration: 50 mM = 0.05 M

Using the formula:

Tm = 81.5 + 0.41 * (50) - (675 / 16) + 16.6 * log10(0.05)

Tm = 81.5 + 20.5 - 42.1875 + 16.6 * (-1.301)

Tm = 102 - 42.1875 - 21.5966

Tm ≈ 38.22 °C

This value provides a starting point for optimizing annealing temperatures in your experiments.