The available technology normally allows 300-500 bases to be read in a sequencing reaction For short DNA sequences, a single set of sequencing reactions is usually adequate. For long DNA sequences, however, the DNA should be cleaved into smaller fragments and such fragments are then sequenced. The four sets of deoxyoligonucleotides are generated by subjecting a purified 3' or 5' end labeled deoxyoligonucleotide to a base-specific chemical reagent that randomly cleaves DNA at one or two specific nucleotides. Since only end-labeled fragments are observed, following autoradiography of the sequencing gel, four DNA ladders are observed. Piperidine then displaces the modified guanine from its sugar.
Formic acid weakens A and G glycosidic bonds by protonating purine ring nitrogens.
The purines can then be displaced by piperidine. The fragments of these bases can then be displaced by piperidine. In the presence of NaCI, only C reacts with hydrazine. The modified C can then be displaced with piperidine. This is the only sequencing method available for small fragments.