Denaturating Electrophoresis in DNA Sequencing: A Brief History and Current Protocols

Two common DNA sequencing methods rely on the principle of measuring relative distances of individual nucleotides from a fixed point in a DNA chain (Sanger and Coulson 1975; Maxam and Gilbert 1977). Base-specific sequencing reactions produce nested sets of fragments, sharing one end and terminating at all positions of a given base, for example guanine, in a DNA chain. The ability to determine a sequence is thus dependent on a separation system which is capable of resolving two DNA fragments that differ in length by a single nucleotide. Until very recently, the only system with adequate resolution, suggested in the 1960s, was based on electrophoresis in a flat polyacrylamide gel with a Tris-borate buffer system (Peacock and Dingman 1967). Electrophoresis was conducted between two glass plates in a simple apparatus, based on the design of Studier (1973). After a certain time, controlled by the mobility of marker dyes, the electrophoresis was terminated and the gel was exposed to an X-ray film or to a phosphor-imaging screen to detect DNA bands labelled with 32P, 35S or 33P (batch technique). The nucleotide sequence was then read from the image using band order and specificity of the sequencing reaction (track identity) (Fig. 1A).