Mutation Detection by PCR, GC-Clamps, and Denaturing Gradient Gel Electrophoresis

Abstract

Denaturing gradient gel electrophoresis (DGGE) allows the separation of DNA molecules differing by as little as a single base change.1-5 The separation is based on the melting properties of DNA in solution. DNA molecules melt in discrete segments, called melting domains, when the *temperature or denaturant concentration is raised. Melting domains vary from about 25 base pairs (bp) to several hundred bp in length, and each melts cooperatively at a distinct temperature called a Tm. Due to the considerable contribution of stacking interactions between adjacent bases on a DNA strand to double helical stability, the Tm of a melting domain is highly dependent on its nucleotide sequence. The Tms of DNA fragments differing by even very small changes, such as a single base substitution, can differ by as much as 1.5°C. In the DGGE system, DNA fragments are electrophoresed through a polyacrylamide gel that contains a linear gradient, from top to bottom, of increasing DNA denaturant concentration. As a DNA fragment enters the concentration of denaturant where its lowest temperature melting domain melts (equivalent to the Tm of the domain), the molecule forms a branched structure that has a retarded mobility in the gel matrix. If the gradient conditions are chosen properly, DNA fragments differing by single base changes begin branching, and hence slowing down, at different positions in the gel, resulting in the separation of the fragments at the end of the electrophoretic run.