dideoxynucleotides

Dideoxynucleotides are nucleotide analogs used to terminate DNA synthesis. Each ddNTP is a deoxynucleoside triphosphate in which the sugar portion lacks both the 2' and 3' hydroxyl groups, yielding 2',3'-dideoxyribose. The four bases—ddATP, ddTTP, ddCTP, and ddGTP—are the standard forms employed in sequencing reactions.

In DNA synthesis, a ddNTP can be incorporated by a DNA polymerase, but the molecule cannot form a subsequent phosphodiester bond because it lacks a 3' hydroxyl group. As a result, once a ddNTP is added, chain elongation stops. This property makes ddNTPs valuable for generating DNA fragments of defined lengths that terminate at specific bases.

Historically, ddNTPs enabled the Sanger dideoxy sequencing method, introduced in the late 1970s by Frederick Sanger and colleagues. In the classic approach, a mixture of normal deoxynucleotides (dNTPs) and small amounts of one ddNTP is used in each reaction, producing a nested set of terminated fragments that can be resolved by size. Modern implementations typically use fluorescently labeled ddNTPs and automated capillary electrophoresis to read the terminated bases, allowing rapid and highly accurate sequence determination.

Today, ddNTPs are mainly associated with traditional Sanger sequencing and are often used for targeted, small-scale sequencing or for validation tasks, rather than high-throughput whole-genome approaches.