Nucleic Acids in Biotechnology 169
Figure 5.1 The principles of classical DNA sequencing. A primer oligodeoxynucleotide is annealed to the DNA tem-
plate to be sequenced (top) and four separate extension reactions are carried out in the presence
of DNA polymerase I, the four deoxynucleoside triphosphates (one usually^32 P-labelled and a single
2 -3-dideoxynucleoside triphosphate) to produce a series of truncated extension products (middle). The
products of the reactions are separated by denaturing polyacrylamide gel electrophoresis and the gel
autoradiographed to obtain a DNA sequence ladder (bottom)
the DNA to become sensitive at these sites to cleavage by alkaline hydrolysis. The fragments created are then
separated by polyacrylamide gel electrophoresis in very much the same way as for Sanger sequencing.
5.1.2 Automated Fluorescent DNA Sequencing
Machines have now been developed to separate and identify the products of dideoxy-sequencing reac-
tions. Here a fluorescent label is built into a set of four alternative dideoxynucleotide chain terminators