ABSTRACT
The chemical method of DNA sequencing has largely been superseded by the method of Sanger, which uses four specific dideoxynucleotides (ddNTPs) to terminate enzymically synthesized copies of a template (Fig. 1b). A sequencing primer is annealed to a ssDNA template molecule and a DNA polymerase extends the primer using dNTPs. The extension reaction is split into four and each quarter is terminated separately with one of the four specific ddNTPs, and the four samples (usually radioactive) are analyzed by PAGE. The dideoxynucleotides act as chain terminators since they have no 3-OH group on the deoxyribose which is needed by the polymerase to extend the growing chain. The label can be incorporated during the synthesis step (e.g. [-35S]dATP) or the primer can first be end-labeled with either radioactivity or fluorescent dyes. The latter are used in some automatic DNA sequencers although it is more common to use fluorescent dideoxynucleotides.
