This equation is inappropriate for probes less than about 50 nucleotides. The number of each particular nucleotide in the probe is inserted into the equation in place of the letters. Melting temperature in solution is determined by plotting O.D versus temperature. The mid point on the S-shaped curve is the melting temperature. Breslauer, et al 3 showed that melting behavior of a DNA duplex is predictable from its primary sequence.
C t is the molar concentration of strands.
The values for D H and D S are shown in the table. The applicability of these equations to laboratory situations varies as additional components in the hybridization environment are altered. It is best to consider these predictions guidelines, since they may vary for particular sequences from empirically derived determinations. Background Problems Moderate background on filter hybridizations is common. Howley, P.M., Israel, M.F., Law, M.F., and Martin, M.A. J. Biol.