The Sanger method for DNA sequencing. (a) DNA synthesis involves a reaction between the 3′-hydroxyl group of the primer dNTP and the phosphate group of an incoming dNTP. (b) The Sanger method uses ddNTPs, which lack the 3′-hydroxyl group, to halt DNA synthesis at a particular nucleotide. (c) In the sequencing reaction, DNA synthesis is carried out with a mixture of dNTPs and a ddNTP to extend a radiolabeled primer. A different ddNTP is used in each reaction. The products are analyzed by autoradiography to determine the nucleotide sequence.