PCR Sequencing

Abstract

The primary property of both PCR and dideoxy DNA sequencing (1) is DNA synthesis. Both techniques require a DNA template and a specific primer to drive a DNA polymerization reaction. Whereas in PCR chain elongation of the polymerization process goes to completion, in DNA sequencing it is continuously interrupted by the presence of 2′–3′ dideoxy derivatives of the natural dNTP precursors. As a consequence, a mixture of single-stranded fragments is generated, called a nested set. In a nested set all fragments have an identical 5′-end whereas the 3′-ends are formed by the specific ddN analogues. Since the 5′-end is provided by the primer, it is of utmost importance to have good quality synthetic oligonucleotides with intact 5′-ends.