chapter  55
2 Pages

DGGE: Denaturing Gel Gradient Electrophoresis

Denaturing gel gradient electrophoresis (DGGE) can be used to detect polymorphisms or very fine mutations of DNA, when even a single base pair is substituted in a DNA fragment. It can be applied to fragments of several hundreds of base pairs and does not require prior knowledge of the polymorphic site. This method is also based on the properties of renaturation of different strands of DNA. However, unlike with SSCP (see Profile 54), the denaturation of the DNA occurs during electrophoresis, not before. For this purpose, the products of amplification are deposited on the gel, which contains either an increasing concentration of a chemical agent denaturing the DNA (urea formamide in the case of DGGE) or a temperature that is increasingly higher during the electrophoresis1 (temperature gel gradient electrophoresis or TGGE). During the electrophoresis, the DNA migrates first to the double strand state, then encounters conditions that denature the least stable molecule with low melting temperature, forming a partly single-stranded structure called "branched" or "ramified". This partial denaturation leads to a reduction of its electrophoretic mobility (the fragment almost stops migrating), so that its filial position in the gel depends exclusively on the melting temperature of the least stable field and of the nucleotide sequence of the latter. It is estimated that around 95% of the sequence variations, even point mutations, of the least stable fusion field can be detected by differences in electrophoretic migration.