We have developed an affinity capillary electrophoresis for gene mutation assay. We show a method for separating a mixture of oligodeoxynucleotides having single-base difference using oligodeoxynucleotide-polyacrylamide conjugate. Because the conjugate behaved similarly to nonionic polyacrylamide in terms of migration rate, it can be used as a pseudoimmobilized affinity ligand in a polyacrylamide-coated capillary. Oligodeoxynucleotide having an oncogene sequence and a version with one base substituted were completely separated by this method. The magnesium-ion concentration was found to be a key factor in achieving efficient separation of oligodeoxynucleotides with the same chain length.