The first method developed for rapid sequence analysis of cloned DNA fragments involved chemical modification and cleavage of specific nucleotides, followed by electrophoresis on high-resolution denaturing acrylamide gels [136, 137]. This chemical sequencing method was developed in 1977 by MAXAM and GILBERT.
DNA polymerases copy single-stranded DNA templates, by adding nucleotides to a growing chain (extension product). Chain elongation occurs at the 3’ end of the primer. The deoxynucleotide added to the extension product is selected by base-pair matching to the template.
The extension product grows by the formation of a phosphodiester bridge between the 3’-hydroxyl group at the growing end of the primer and the 5’-phosphate group of the incoming deoxynucleotide [138]. The growth is in the 5’→3’ direction.
DNA polymerases can also incorporate analogues of nucleotide bases. The dideoxy method of DNA sequencing developed by SANGER et al. [139, 140] takes advantage of this ability by using 2’,3’-dideoxynucleotides as substrates. When a dideoxynucleotide is incorporated at the 3’ end of the growing chain, chain elongation is terminated selectively at A, C, G, or T because the chain lacks a 3’-hydroxyl group.
© 2001 Alexander Binder