We have developed a method to produce a set of four duplex oligonucleotides, each with a different labeled base at a given position, from one template-primer combination. The template oligonucleotide is synthesized with a mixture of all four bases at the position of interest, and the primer oligonucleotide hybridizes to the template at all bases 3' from the position of interest. Specifically labeled substrates are then produced by differential incorporation of each of the four labeled nucleotides in four separate reactions. This method is more cost-effective than synthesizing four separate duplex oligonucleotides with different base pairs at the position of interest. We have successfully used this method to test nucleotide substitutions at several positions of a DNA recognition site for the phage T4 type II DNA topoisomerase.