Denaturing Gradient Gel Electrophoresis (DGGE)

How does DGGE work?

Denaturing gradient gel electrophoresis (DGGE) separates amplified 16S rRNA genes using a gel containing chemicals which break apart the strands of the DNA molecule. Because the 16S gene segments are the same length, separation must be made based on the genetic sequences rather than on size. Denaturing gradient gels have a gradient of increasing concentration (top to bottom) of denturing chemicals. Separation of 16S rRNA genes from different bacteria can be achieved according to the ease with which the double helix is separated, and this in turn is determined by the unique base sequence of the gene (see below).

Four nucleotides (A, T, G and C) make up the DNA molecule. The order and amount of the nucleotides determines how much denaturant is required to break apart the two strands of the DNA fragment. In the DNA double helix, an "A" on one strand pairs with a "T" on the opposite strand; while "G" pairs with "C". An A:T base pair shares two hydrogen bonds; a G:C base pair shares three bonds and as such, the G:C bond is more difficult to separate. The higher the concentration of G:C bonds within a molecule of DNA, the more denturant required to separate the strands.

Amplified DNA is loaded at the top of the denturing gel, and the DNA fragments move down the gel (in response to electrical charge) into increasing concentrations of chemical denaturant. Depending on the base sequence, the DNA separates from a linear molecule to a Y-shaped or T-shaped molecule, which slows its migration in the gel matrix due to increased surface area (similar to an opened parachute). DNA fragments from different species will denature at different places in the gradient gel because of differences in DNA sequences and the amount of chemical denaturant required to achieve separation. Identical sequences will migrate to the same area of the gel creating a band within the gel. Individual bands are then excised for sequencing and the sequences obtained are placed in a phylogenetic lineage, according to sequences recorded in the database.

MI has developed ways to use DGGE to specifically test for sulfate reducing bacteria (DGGE-SRB) and also Dehalococcoides (DGGE-DHC), by using primers that isolate the 16S DNA from these organisms, excluding DNA from non-target organisms. Additionally, DGGE can be used to characterize the presence of fungi (DGGE-FGI) by using the 28S rRNA gene, which is present in fungi, rather than 16S gene which is present in prokaryotes.