Tm DNA
- Created by
- Renato Passos, Eng. de Software
- Reviewed by
- Renato Passos, Eng. de Software
Last updated: Apr 18, 2026
Formula
Tm = 64.9 + 41·(GC-16.4)/L
About this calculator
The DNA Tm calculator estimates the melting temperature of a DNA sequence, indicating when strands separate. It uses the formula Tm = 64.9 + 41·(GC-16.4)/L, where GC is the number of G-C base pairs and L is the total length. This formula accounts for G-C stability from double hydrogen bonds and sequence length effects.
Melting temperature is critical in PCR and hybridization experiments. Sequences with more G-C pairs have higher Tm due to stronger bonding. Shorter DNA strands destabilize more easily. The formula balances these factors to predict realistic lab values.
Use this calculator to design primers, optimize PCR reactions, or analyze genetic sequences. Adjusting Tm ensures primers bind specifically to targets. However, this simplified calculation doesn't account for salt concentration or other reaction conditions.
Cautions: the formula is an approximation for double-stranded DNA under ideal conditions. For extreme accuracy, experimental methods or complex formulas (like Wallace) are recommended. Sequences with repeats or non-uniform composition may yield less reliable results.
Frequently asked questions
Why does the formula subtract 16.4 from GC?
The 16.4 value is an empirically adjusted normalization factor to balance the G-C pair stability relationship. It's not a direct physical constant but improves accuracy for typical sequences.
Does this calculator work for RNA?
No, the formula is DNA-specific. RNA has different bonding types and secondary structures requiring specialized calculation methods.
How does ambient temperature affect the result?
Ambient temperature doesn't impact the theoretical calculation, which predicts Tm under standardized conditions (usually 50°C, 0.1M NaCl). Real lab variations may occur.
Should I consider salt concentration?
This simplified formula ignores salts, ions, and other factors. For critical applications, use software that includes corrections for Mg²⁺ concentration and other effects.