(a) A single strand of the DNA to be sequenced (blue line) is hybridized to a 5′-end-labeled synthetic deoxyribonucleotide primer. The primer is elongated in four separate reaction mixtures containing the four normal deoxyribonucleoside triphosphates (dNTPs) plus one of the four dideoxyribonucleoside triphosphates (ddNTPs) in a ratio of 100 to 1. A ddNTP molecule can add at the position of the corresponding normal dNTP, but when this occurs, chain elongation stops because the ddNTP lacks a 3′ hydroxyl. In time, each reaction mixture will contain a mixture of prematurely terminated chains ending at every occurrence of the ddNTP (yellow). (b) Three of the labeled chains that would be generated in the presence of ddGTP from the specific DNA sequence shown in blue. (c) An actual autoradiogram of a polyacrylamide gel in which more than 300 bases can be read. Each reaction was carried out in duplicate using Sequenase™, a commercial preparation of the DNA polymerase from bacteriophage T7. [Part (c) courtesy of United States Biochemical Corporation.]