ddATP

ddATP, short for dideoxyadenosine triphosphate, is a dideoxynucleotide used as a chain-terminating substrate in DNA sequencing, notably the Sanger method. It differs from the regular dATP by lacking the 3' hydroxyl group on the deoxyribose sugar, which prevents the formation of the next phosphodiester bond and thus halts DNA synthesis after its incorporation. When a DNA polymerase incorporates ddATP opposite a template thymine, the growing DNA strand cannot be extended further.

In traditional Sanger sequencing, small amounts of ddATP are included with the standard deoxynucleoside triphosphates (dNTPs). Each of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) can terminate a chain at its corresponding base. By using labeled ddNTPs, sequencing reactions generate fragments that end at every instance of a given base. Separation by size (via gel or capillary electrophoresis) and detection of the labels yields a readable sequence of the newly synthesized strand. Modern fluorescence-based Sanger methods typically use a mixture of four differently labeled ddNTPs to enable simultaneous detection of all termination events.

ddATP is one of the four chain-terminating nucleotides and is used in contexts where a thymine in the template directs incorporation of adenine in the growing strand. Although largely superseded for routine sequencing by next-generation methods, Sanger sequencing with ddNTPs remains a foundational technique in molecular biology and is still employed for targeted, high-accuracy reads and validation experiments.

See also: Sanger sequencing, ddNTP, dNTP, chain termination.