Dideoxyribonucleotide Triphosphates

Termination occurs because the incorporated ddNTP lacks a 3′-hydroxyl group. DNA polymerase therefore cannot form the next phosphodiester bond with an incoming nucleotide. The chain stops precisely at the position where that modified building block was added, creating a fragment whose endpoint records a specific location in the template sequence.

DNA polymerase incorporates a ddNTP when its base pairs with the complementary base in the template. This pairing restricts incorporation to the corresponding position in the growing strand. Because extension then stops, each terminated fragment links a particular endpoint to the complementary template information, supporting sequence determination after the fragments are analyzed.

Both types can serve as DNA polymerase substrates when they pair appropriately with the template, but their consequences differ after incorporation. A deoxyribonucleotide retains the hydroxyl groups needed for continued extension, whereas a ddNTP lacks both the 2′- and 3′-hydroxyl groups. That structural difference converts incorporation into a chain-termination event.

Controlled incorporation generates a collection of DNA fragments that terminate at different positions. The distribution of fragment endpoints reflects where complementary bases were added during synthesis. Separating and detecting these fragments reveals the order of positions along the newly synthesized strand, which allows the template sequence to be determined.

The process requires a DNA template, DNA polymerase, and ddNTPs that can be incorporated during complementary strand synthesis. Incorporation produces terminated fragments of different lengths. Researchers then separate and detect those fragments, using their endpoints to reconstruct the template sequence rather than examining the intact template directly.

Fragment separation distinguishes products according to where synthesis stopped, while detection identifies the sequence information associated with those endpoints. Reading the ordered termination pattern makes it possible to infer the template sequence. The method therefore converts a biochemical event, polymerase-mediated termination, into an experimentally interpretable sequence result.

Their chain-termination behavior supports gene analysis, mutation identification, and molecular diagnostics. In each application, the central measurement is the sequence inferred from terminated DNA fragments. Biochemically, the method connects nucleotide structure, template-directed polymerase activity, and phosphodiester-bond formation to practical analysis of genetic information.