The separation principle of denaturing gradient gel electrophoresis (DGGE) is based on the melting (denaturation) properties of DNA in solution. DNA molecules melt in discrete segments called melting domains, when the temperature or denaturant concentration is raised. The melting temperature (Tm) of a melting domain depends on its nucleotide sequence. As DNA fragments are electrophoresed through a linear gradient of increasing denaturing concentration, the separation of double-stranded DNA into single-stranded segments increases. This causes the DNA segment to form a less uniform three-dimensional structure that moves through a polyacrylamide matrix at a reduced rate. The gradient itself can be arranged in either a perpendicular or a parallel orientation relative to the electric field, depending on whether a broad or a narrow range of denaturing conditions is required.

As a matter of practice, a perpendicular gradient gel, where the gradient is perpendicular to the electric field, is typically composed of a broad denaturing gradient range, often spanning anywhere from 0 to 100%. In parallel DGGE the denaturing gradient is parallel to the electric field, and the range of denaturant is somewhat narrower, so fragments can separate from one another more readily in a given distance.

As a DNA fragment enters the concentration of denaturant where its lowest temperature domain melts, the molecule begins branching and, hence, slowing down at a unique position in the gel. This results in separation of different fragments at the end of the run. The attachment of a GC-rich segment, called a GC clamp, which never denatures at the conditions chosen for the experiment, allows for a branched-shaped molecule whose shape is anchored as a double-stranded molecule by the GC clamp. Using this strategy, detection of almost all single-base changes in the fragment can be analyzed. To determine the theoretical melting domains of the DNA fragment of interest, a computer program developed by Lerman is typically used.

Although DGGE can require significant preparation and analysis time compared with similar techniques such as SSCP, it offers the unique benefit of not having to actually sequence, prepare, or test primers for the DNA segment to be studied. Still, the rate of polymorphism detection is somewhat lower than with SSCP (Sheffield et al., 1993). Similar techniques related to DGGE include genomic denaturing gradient gel electrophoresis (gDGGE) (Burmeister et al., 1991). In this method restricted genomic DNA is run in denaturing gradient gels and then transferred to a nylon membrane; sequences of interest are detected by standard hybridization methods much like RFLP.

References

Burmeister, M., diSibio, G., Cox, D. R., and Myers, R. M. Identification of polymorphisms by genomic denaturing gradient gel electrophoresis: application to the proximal region of human chromosome 21. Nucleic Acids Res 19(7): 1475–81, 1991.

Sheffield, V. C., Beck, J. S., Kwitek, A. E., Sandstrom, D. W., and Stone, E. M. The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions. Genomics 16(2): 325–32, 1993.