dideoksynukleotid

A dideoxynucleotide, often abbreviated as ddNTP, is a modified nucleotide that is crucial in molecular biology techniques, particularly DNA sequencing. Unlike a standard deoxynucleotide, a dideoxynucleotide lacks a hydroxyl group at the 3' carbon of its sugar ring. This seemingly small structural difference has significant implications for DNA synthesis.

During DNA replication, the enzyme DNA polymerase adds new nucleotides to a growing DNA strand by forming a phosphodiester bond between the 3' hydroxyl group of the last nucleotide and the 5' phosphate group of the incoming nucleotide. Since dideoxynucleotides lack this 3' hydroxyl group, they cannot be incorporated into a growing DNA chain in this manner. If a dideoxynucleotide is added to the chain, DNA synthesis is terminated at that point because no further nucleotides can be added.

This chain-terminating property is the basis of the Sanger sequencing method, also known as the dideoxy sequencing method. In this technique, DNA is replicated in the presence of a mixture of standard deoxynucleotides and a small amount of each of the four dideoxynucleotides (ddATP, ddTTP, ddGTP, and ddCTP), each labeled with a different fluorescent marker. When a dideoxynucleotide is incorporated into a DNA strand, replication stops. By controlling the ratio of deoxynucleotides to dideoxynucleotides, fragments of varying lengths are generated, each ending with a specific dideoxynucleotide. The fluorescent labels allow for the identification of the terminal dideoxynucleotide at each position, revealing the sequence of the original DNA.