Paul Zoller and Theresa Redila-Flores
Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA
Denaturing gradient gel electrophoresis (DGGE) is one of several methods that can be used to screen DNA fragments for small sequence changes or point mutations. In DGGE, the denaturing environment is created by a combination of uniform temperature and a linear denaturant gradient formed with urea and formamide (Fischer and Lerman 1983). A 100% denaturant solution consists of 7 M urea and 40% formamide. The denaturing gradient may be formed perpendicular or parallel to the direction of electrophoresis. When running a denaturing gradient gel, both the amplified mutant and wildtype DNA fragments are run on the same gel. This way, one can detect a mutation by seeing a band shift between the mutant and wild-type samples.
The separation principle of DGGE is based on the melting behavior of DNA molecules. In a denaturing gradient acrylamide gel, double-stranded DNA is subjected to a denaturant environment and will melt in discrete segments called melting domains. The melting temperature (Tm) of these domains is sequence-specific. When the Tm of the lowest domain is reached, the DNA will become partially melted, creating branched molecules. Partial melting reduces the DNAs mobility in a polyacrylamide gel. Since the Tm of a particular melting domain is sequence-specific, the presence of a mutation will alter the melting profile when compared to the wild-type DNA. Thus, the DNA containing the mutation will have a different mobility compared to the wild type.
In this experiment, we show that parallel DGGE analysis on