Didesoxynukleotids

Didesoxynukleotids, often abbreviated as ddNTPs, are modified nucleotides that play a crucial role in molecular biology, particularly in DNA sequencing. Unlike natural deoxynucleotides (dNTPs), ddNTPs lack a hydroxyl group at the 3' carbon of the deoxyribose sugar. This seemingly small structural difference has profound implications for DNA synthesis. When a ddNTP is incorporated into a growing DNA chain by a DNA polymerase, the absence of the 3'-hydroxyl group prevents the formation of a phosphodiester bond with the next incoming nucleotide. Consequently, DNA chain elongation terminates at that point.

This chain-terminating property is the basis of the Sanger sequencing method, also known as the dideoxy method. In this technique, DNA replication is carried out in the presence of both dNTPs and a small amount of ddNTPs. Each of the four types of ddNTPs (ddATP, ddTTP, ddCTP, and ddGTP) is labeled with a distinct fluorescent marker. By performing four separate reactions, each with a different ddNTP, or by using a single reaction with differentially labeled ddNTPs, fragments of DNA are generated that terminate at every possible position where that specific ddNTP was incorporated. When these fragments are separated by size, typically using gel electrophoresis or capillary electrophoresis, the order of the fluorescent labels reveals the sequence of the original DNA template. While Sanger sequencing is now largely supplanted by next-generation sequencing methods for large-scale projects, ddNTPs remain an important tool in research and diagnostics.