Molecules in a mixture can be separated according to size by electrophoresis, a technique dependent on the fact that dissolved molecules in an electric field move at a speed determined by their charge-mass ratio. Many successful variations of electrophoresis are in general use; the separation of small molecules, such as amino acids and nucleotides, is one example.
Nucleic acids in solution generally have a negative charge because their phosphate groups are ionized; thus they migrate toward a positive electrode. However, nucleic acid molecules consisting of long chains have almost identical charge-mass ratios, whatever their length, because each residue contributes about the same charge and mass. Therefore, if the electrophoresis of nucleic acids were simply carried out in solution, little or no separation of molecules of varying lengths would occur.
Molecules are now most commonly subjected to electrophoresis in a gel, rather than a liquid solution. The size of the pores in such gels limits the rate at which molecules can move through them. Nucleic acids with identical charge-mass ratios separate according to length, with the longer ones moving more slowly. Even very long nucleic acids (chains containing 10,000 to 20,000 residues) that differ in length by only a few percentage points can be separated. In mixtures containing chains of 500 nucleotides or less (and separated on polyacryl amide gels), each chain length can be resolved, which made DNA sequencing possible.