A new rapid method for DNA sequence analysis has been devised. In this method, base-specific cleavage is achieved at partially substituted ribonucleotides which are introduced by DNA polymerase extension in the presence of Mn2+. Access to a target sequence and label incorporation are achieved by extending a restriction fragment primer with DNA polymerase I. After a short initial incorporation with [α-32P]deoxynucleotide triphosphates to label the 5′ region of the target sequence, the triphosphates are removed and the reaction mixture is divided four ways for a second primed extension. The second extension is a cold chase in the presence of Mn2+, all four deoxynucleotides and one of the four ribonucleotides under conditions that result in about 2% ribonucleotides substitution at each position. After cleavage at the restriction site and alkali cleavage at the positions of partial ribosubstitution, each reaction mixture is analysed by electrophoresis on a high-resolution denaturing acrylamide gel. As in the other rapid DNA sequencing methods the extent of DNA sequence that can be determined from a single experiment is limited only by the resolution of the analysing gels. At present some 100 nucleotides of sequence can be determined from a single priming reaction.