dideoksynukleotider

Dideoksynukleotider, often abbreviated as ddNTPs, are a class of nucleotide analogs that play a crucial role in molecular biology, particularly in DNA sequencing. Unlike standard deoxynucleotides (dNTPs), dideoxynucleotides lack a hydroxyl group at the 3' position of their deoxyribose sugar. This seemingly small structural difference has significant implications for DNA synthesis.

During DNA replication or polymerase chain reaction (PCR), DNA polymerase enzymes add new nucleotides to a growing DNA strand by forming a phosphodiester bond between the 3'-hydroxyl group of the existing strand and the 5'-phosphate group of the incoming nucleotide. Because ddNTPs lack this 3'-hydroxyl group, once a dideoxynucleotide is incorporated into a DNA strand, no further nucleotides can be added. This effectively terminates the elongation of that particular DNA chain.

This chain-terminating property is the basis of the Sanger sequencing method, also known as the dideoxy sequencing method. In Sanger sequencing, a reaction mixture contains DNA polymerase, primers, dNTPs, and a small amount of ddNTPs, each labeled with a different fluorescent dye. When the polymerase encounters a ddNTP, synthesis stops at that point. By running this reaction with each of the four ddNTPs (ddATP, ddTTP, ddCTP, ddGTP) separately or together with distinct labels, fragments of varying lengths are produced, each ending with a specific ddNTP. Analyzing the sizes and fluorescent signals of these terminated fragments allows for the determination of the DNA sequence.