Melting Temperature

Every double-stranded molecule has a temperature at which the two strands will separate. This is called the Melting Temperature (T_m) or Annealing Temperature of the molecule. For now we will ignores effects of the concentrations of molecules such salt - which are very important when manipulating DNA in the lab - and some subtle features of DNA structure that we are not dealing with at this level (such as base-stacking rules) and and stress three important factors.

Overall, the melting temperature of a double stranded molecule is:

• A function of Length
• A function of % GC
• A function of % Mismatches

Although we refer to complementarity of two single-stranded nucleic acids, two strands need not be 100% complementary to form a double stranded molecule. In some cases a double stranded molecule will have mismatches which are base pairs with non-Watson-Crick pairings, such as A-G. As long as enough bases are complementary the molecule will be stable. However, the degree of mismatches that are acceptable depends on a number of factors. What is important is that there is sufficient energy in the Hydrogen bonds that do form. This will be a function of length, (which will obviously affect the number of bonds), and also the percentage of base-pairings that are G-C since they are stronger.