Hi everyone,
I need to calculate which fraction (percent) of a given oligo is annealed at given temperature (like 4°C, 10°C, 20 °C and 37°C ).
I found the formula to calculate the Tm (50% annealed):
Tm (°C) = 81.5 + 0.41(%GC) - (675/N)
where %GC is the percentage of G and C and N is the number of nucleotides in the oligo.
Hi Emmanuele,
good question but hard to answer without trying. your formula is incomplete, try with this one and tell me if it helps.
Tm= 81.5 + 0.41x(%GC) - 675/N - (%mismatch)
where % mismatch is 100x
best Xmas
fred
Dear Emmanuele,
you can simulate DNA melting using uMelt Batch software:
https://www.dna.utah.edu/umelt/umb.php
However, melting experiments would give more accurate data (because of precise hybridization conditions like buffer composition and DNA concentration).
Best wishes
I will have a try and hopefully someone can confirm or correct this assumption but having seen Pawel's suggestion it looks a better practical solution given the reliance of these calculations on salt concentrations
If we assume a single binding state not a complex partial melt situation for A+B oligos in equilibrium with AB the annealed state A + B < == > AB
Then the equilibrium constant K is given by .K = [A][B]/[AB]
Then the relation between free energy, ΔG, and K is ΔG° = -RTln K, where R is the ideal gas law constant, and T is the kelvin temperature of the reaction.
A], [B], and [AB] will be equal, and equal to half the initial concentration of double-stranded nucleic acid, [AB]initial. This gives an expression for the melting point of a nucleic acid duplex of
Tm = - ΔG°/Rln ([AB]initial/2)
Delta G can be obtained for all oligos from various manufacturers websites using the nearst neighboar calculation and I suggest that the value of Tm for concentrations other than equimolar can be calculated by substituting the percentage melting in place of the value 2 in the above equation .
References and more detailed explanation can be seen here
https://en.wikipedia.org/wiki/Nucleic_acid_thermodynamics
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