Factors affecting the melting temperature:
(1) the nucleic acids which are hybridized, with RNA-RNA hybrids more stable than RNA-DNA hybrids, which are more stable than DNA-DNA hybrids
(2) the length of the probe, with longer probes being more stable
(3) the base composition of the probe, with higher GC content more stable
(4) the extent of sequence identity between probe and target, with closer matches being more stable
(5) the concentration of monovalent cations, with probes more stable at higher concentrations
(6) the concentration of formamide, with probes more stable at lower concentrations
DNA-DNA hybrids 11-22 base pairs
Tm in °C =
= (4 * (number of G+C residues)) + (2 * (number of A+T residues))
DNA-DNA hybrids more than 22 base pairs
Tm in °C =
= 81.5 + (16.6 * (log10 (molarity of monovalent cations))) + (0.41 * (percent GC content of probe as a whole number) - (500 / (length of probe in bases)) - (0.62 * (percent formamide as a whole number))
RNA-RNA hybrids
Tm in °C =
= 79.8 + (18.5 * (log10 (molarity of monovalent cations))) + (0.58 * (percent GC content of probe as a whole number) + (0.0012 * ((percent GC content of probe as a whole number) ^ 2)) - (820 / (length of probe in bases)) - (0.35 * (percent formamide as a whole number))
RNA-DNA hybrids
Tm in °C =
= 79.8 + (18.5 * (log10 (molarity of monovalent cations))) + (0.58 * (percent GC content of probe as a whole number) + (0.0012 * ((percent GC content of probe as a whole number) ^ 2)) - (820 / (length of probe in bases)) - (0.5 * (percent formamide as a whole number))
Temperature of Hybridization
hybridization temperature in °C=
= Tm - 25
