DNA Melting Temperature (Tm) Calculator
Calculate the melting temperature of DNA primers and oligonucleotides.
Supports both basic (Wallace rule) and nearest-neighbor thermodynamic methods.
What Is Melting Temperature (Tm)? The melting temperature of a DNA molecule is the temperature at which 50% of the DNA strands are in double-helix form and 50% are single-stranded (denatured). It is one of the most important parameters in molecular biology, especially for designing PCR (Polymerase Chain Reaction) primers.
Why Tm Matters In PCR, primers must bind specifically to their target DNA. If the annealing temperature is too high, primers will not bind. If too low, primers may bind nonspecifically to wrong sequences. The optimal annealing temperature is typically 3-5 degrees Celsius below the Tm of the primers.
The Basic Formula (Wallace Rule) For short DNA sequences (under 14 base pairs): Tm = 2(A+T) + 4(G+C) degrees Celsius. Where A, T, G, C are the counts of each nucleotide. This formula accounts for the fact that G-C base pairs have 3 hydrogen bonds (stronger) while A-T base pairs have only 2.
The Salt-Adjusted Formula For longer sequences: Tm = 81.5 + 16.6 * log10([Na+]) + 41 * (GC fraction) - 675/N. Where [Na+] is the sodium ion concentration (typically 0.05 M for standard PCR), GC fraction is the proportion of G and C bases, and N is the sequence length. This accounts for ionic strength effects on DNA stability.
GC Content and Stability DNA sequences with higher GC content have higher melting temperatures because G-C pairs form three hydrogen bonds versus two for A-T pairs. The GC content of a primer is ideally between 40-60% for optimal PCR performance.
Practical PCR Guidelines Primers should typically be 18-25 bases long. The Tm of forward and reverse primers should be within 5 degrees of each other. Primer GC content between 40-60% is ideal. The last 5 bases at the 3’ end should contain no more than 2 G or C bases (GC clamp) to avoid strong nonspecific binding.