A technique for determination of the base sequence of a homogeneous polynucleotide that acts as a template in a replication system. A small amount of a specific dideoxynucleoside triphosphate is included with the four normal deoxynucleotide triphosphates during replication. Chain termination occurs whenever a dideoxynucleotide is incorporated that generates a labelled polynucleotide whose chain length, evaluated by polyacrylamide-gel electrophoresis, is indicative of the distance from the 5'-end of the primer to the position at which the normal nucleotide occurs. Detection of the electrophoresis bands may be by a label such as ³²P or, more recently, by a fluorophore incorporated into the primer (dye primer sequencing) or the dideoxynucleotide (dye terminator sequencing). In fact, in a variation of the dye terminator method, the labelling of each of the four dideoxynucleotides with a fluorophore that emits at a different wavelength permits the identification of the base that terminates each electrophoresis band within a separated mixture of all four (single lane sequencing).