The melting behaviour of a DNA fragment carrying the mouse βmajglobin promoter was investigated as a means of establishing procedures for separating fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lermen (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp. to a 135 bp DNA fragment carrying the mouse β-globin.promoter significantly alters the pattern of melting within the promoter When the promoter, is attathed to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agre with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels. © 1985 IRL Press Limited.