Denaturing Gradient Gel Electrophoresis to Detect Methylation Changes in DNA

Denaturing gradient gel electrophoresis (DGGE) is a technique that fractionates DNA molecules on the basis of their melting behavior and thereby permits the separation of DNA fragments with local variations in base composition. The separation of DNA fragments by DGGE is determined by the nucleotide sequence, rather than size. This approach is effective when part of the molecule is relatively dense in G+C pairs. This separation is possible because of the pronounced drop in electrophoretic mobility in a polyacrylamide gel that occurs when a region of a DNA molecule melts, thereby forming a structure that is partly helical and partly random chain. The electrophoretic mobility of these partly melted DNA fragments is much lower than that of fully helical or fully dissociated molecules. The low residual mobility of the fragment restricts migration into more strongly denaturing regions of the gradient gel and results in focusing of the band. This property can be applied to detect the difference in melting temperature between methylated and nonmethylated DNA fragments after chemical treatment, or to enrich genomic regions in which aberrant methylation occurs. In this chapter, the application of DGGE to the analysis of genomic DNA methylation is reviewed.