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Tm (Wallace Rule, short oligos)
60.0 °C
Tm (Wallace Rule, short oligos) vs Oligonucleotide Length
सूत्र
## DNA Melting Temperature The melting temperature (Tm) is the temperature at which 50% of double-stranded DNA molecules are denatured into single strands. ### Wallace Rule (for short oligos < 20 bp) **Tm = 2(A+T) + 4(G+C)** °C ### Marmur-Doty (for longer DNA) **Tm = 64.9 + 41 × (G+C - 16.4) / N** °C GC base pairs have three hydrogen bonds (vs two for AT), making them more thermally stable.
हल किया गया उदाहरण
A 20-mer with 10 GC bases and 10 AT bases.
- 01AT count = 20 - 10 = 10
- 02Wallace: Tm = 2(10) + 4(10) = 20 + 40 = 60°C
- 03GC content = 10/20 × 100 = 50%
अक्सर पूछे जाने वाले प्रश्न
When should I use each formula?
The Wallace rule is best for short oligonucleotides (< 20 bp) like PCR primers. The Marmur-Doty and nearest-neighbor thermodynamic methods are better for longer sequences. Salt concentration and mismatches also affect Tm.
How does salt affect Tm?
Higher salt concentration stabilizes DNA by neutralizing backbone charges, increasing Tm. A common correction: Tm increases by about 16.6°C per log10 increase in [Na+].
Why is Tm important for PCR?
PCR annealing temperature is typically set 3-5°C below the Tm of the primers. If the annealing temperature is too high, primers do not bind; too low, and nonspecific binding occurs.
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