A method for detection of single base substitutions in DNA fragments. Putative mutant and normal double-stranded DNA fragments are applied along one edge of an agarose gel slab that contains a denaturing agent (e.g. urea and formamide) in a gradient perpendicular to the direction of electrophoresis. At a low concentration of the denaturant the fragments are more mobile, and at high concentration they are unfolded and therefore less mobile. The electrophoretic band describes a sigmoid curve on the developed gel; the denaturant concentration represents the mid-point of the curve and is characteristic of the sequence. Introduction of a GC-rich sequence, which is very stable to denaturation and thus acts as a GC clamp, into the DNA allows detection of mutations in the more stable regions of the DNA.