ddntp

ddNTP stands for dideoxynucleoside triphosphate, a chain-terminating nucleotide used in the Sanger method of DNA sequencing. These molecules are analogs of standard deoxynucleoside triphosphates (dNTPs) but lack both the 2' and 3' hydroxyl groups on the sugar, rendering them unable to form a phosphodiester bond with the next nucleotide after incorporation.

In a sequencing reaction, DNA polymerase incorporates ddNTPs opposite the template according to the base to be added. Because each ddNTP lacks the 3' hydroxyl, once a ddNTP is incorporated, further extension stops. A mixture of four ddNTPs with the normal four dNTPs produces a population of DNA fragments ending at every occurrence of each terminator base. The four commonly used ddNTPs are ddATP, ddTTP, ddCTP, and ddGTP.

Detection is achieved by labeling the ddNTPs; in modern protocols each ddNTP carries a distinct fluorescent dye, allowing a single reaction to generate a color-encoded ladder read by capillary electrophoresis or automated sequencers. Earlier methods used radioactive or differently labeled nucleotides and observed fragment lengths on polyacrylamide gels. The ratios of ddNTPs to dNTPs and the overall concentrations are carefully balanced to create a readable distribution of termination events, producing a readable sequencing ladder.

ddNTPs were a key breakthrough in the development of Sanger sequencing, introduced by Frederick Sanger in 1977. They remain standard in traditional capillary Sanger sequencing for short to medium-length reads and in educational settings, although newer high-throughput platforms employ different sequencing chemistries, such as reversible terminator nucleotides in sequencing-by-synthesis methods.