Dideoxyribonucleoside triphosphates
Dideoxyribonucleoside triphosphates ( ddNTP ) are artificial DNA nucleotides that are used in DNA sequencing according to Sanger . The ddNTPs are structured like the deoxyribonucleoside triphosphates (dNTPs). However, the ribose (sugar) at position 2 'and 3' is deoxidized. As a result, the 3'-carbon atom lacks the hydroxyl group to which the next nucleotide is attached during polymerization.
If the DNA polymerase incorporates such a ddNTP at one point during replication, no further nucleotide can be added and the polymerization stops at this point. One can therefore simply speak of stop nucleotides.
In DNA sequencing according to Sanger , the four natural dNTPs and one of the four artificial ddNTPs are now added to a PCR mixture with the DNA to be sequenced and a corresponding primer . The polymerase now occasionally incorporates a ddNTP in a random distribution, which stops the polymerization. The result is a mixture of pieces of the DNA to be sequenced of different lengths. This experiment is carried out with all four ddNTPs and the four approaches are applied next to one another in a polyacrylamide gel electrophoresis . The sequence of the bases on the DNA segment can then be read from the pattern of the resulting bands.
Individual evidence
- ↑ Sanger, F. et al. (1977): DNA sequencing with chain-terminating inhibitors. In: Proc. Natl. Acad. Sci. USA 74 (12), pp. 5463-5467; PMID 271968 ; PMC 431765 (free full text, PDF).