Automated DNA sequencing

Abstract

Abstract DNA sequencing has undergone great advances since the initial developments of Maxam and Gilbert (1) and Sanger et al (2) in the 1970s. The Maxam“Gilbert or chemical method first employs specific chemicals to modify DNA at one or two bases. The DNA is then cleaved at the modified site to generate fragments shorter by one base than the targeted base. In the Sanger or dideoxy method, a specific primer is annealed and enzymatically extended to synthesize a complement of the unknown sequence. The reaction mixtures contain the four deoxynucleoside triphosphates (dNTPs) plus one dideoxynucleoside triphosphate (ddNTP). Incorporation of the dideoxy analogue inhibits further extension of the synthesis. By carefully titrating the reagents and substrate, both methods ensure that every possible base position is the site of either cleavage or strand elongation termination. Therefore, a complete nested set of fragments is generated. Both methods use high resolution polyacrylamide gel electrophoresis to separate these fragments. Until recently, radioactive markers were the only method of labelling these fragments, with autoradiography the method of detection. Sets of reactions are loaded onto adjacent gel lanes, one reaction per lane, and the sequence is determined by reading bands going up the autoradiograph. In this maimer 300 bases or more can be determined from a given, fragment.