20
10
10
50
%
60
°C
51.8
°C
20
10
10
50
%
60
°C
51.8
°C
The DNA Melting Temperature (Tm) Calculator estimates the temperature at which 50% of a DNA duplex dissociates into single strands. Knowing the Tm is critical for designing PCR primers, setting hybridization temperatures, and optimizing probe-based assays. This calculator provides two common estimation methods: the Wallace rule for short oligonucleotides (≤14 bases) and the basic Tm formula for longer sequences.
Two formulas are provided:
Wallace Rule (for oligos ≤14 nt):
Tm = 2(A + T) + 4(G + C)
This simple formula assigns 2°C per A-T base pair and 4°C per G-C base pair.
Basic Formula (for longer sequences):
Tm = 64.9 + 41 × (G+C − 16.4) / N
Where N is the total number of bases. This formula accounts for the stabilizing effect of GC content on the duplex.
Note: For the most accurate Tm calculations, nearest-neighbor thermodynamic methods should be used, but these simple formulas provide good estimates for primer design.
Inputs
Results
A 20-nucleotide primer with equal base composition (50% GC) has a Wallace Tm of 60°C and a basic Tm of approximately 56.7°C.
Inputs
Results
An 18-nucleotide primer with 66.7% GC content has a basic Tm of about 62.1°C, reflecting the higher stability from GC base pairs.
The Wallace rule (Tm = 2(A+T) + 4(G+C)) is most accurate for short oligonucleotides of 14 bases or fewer. For longer sequences (15–70 bases), the basic formula gives a better estimate. For the highest accuracy, especially with mismatches or modified bases, use nearest-neighbor thermodynamic calculations.
Ideal PCR primers should have a Tm between 55°C and 65°C, with an optimal around 60°C. Both primers in a pair should have Tm values within 2–3°C of each other to ensure they anneal at similar temperatures during PCR cycling.
Guanine-cytosine (G-C) base pairs form three hydrogen bonds, while adenine-thymine (A-T) pairs form only two hydrogen bonds. The additional hydrogen bond makes G-C pairs more thermodynamically stable, requiring more energy (higher temperature) to dissociate the duplex.
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