Temperature Gradient Gel Electrophoresis - Denaturing Gradient Gel Electrophoresis

Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) works by applying a small sample of DNA (or RNA) to an electrophoresis gel that contains a denaturing agent. Researchers have found that certain denaturing gels are capable of inducing DNA to melt at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components, even those as small as 200-700 base pairs.

What is unique about the DGGE technique is that as the DNA is subjected to increasingly extreme denaturing conditions, the melted strands fragment completely into single strands. The process of denaturation on a denaturing gel is very sharp: "Rather than partially melting in a continuous zipper-like manner, most fragments melt in a step-wise process. Discrete portions or domains of the fragment suddenly become single-stranded within a very narrow range of denaturing conditions" (Helms, 1990). This makes it possible to discern differences in DNA sequences or mutations of various genes: sequence differences in fragments of the same length often cause them to partially melt at different positions in the gradient and therefore "stop" at different positions in the gel. By comparing the melting behavior of the polymorphic DNA fragments side-by side on denaturing gradient gels, it is possible to detect fragments that have mutations in the first melting domain (Helms, 1990). Placing two samples side-by-side on the gel and allowing them to denature together, researchers can easily see even the smallest differences in two samples or fragments of DNA.

There are a number of disadvantages to this technique: "Chemical gradients such as those used in DGGE are not as reproducible, are difficult to establish and often do not completely resolve heteroduplexes" (Westburg, 2001). These problems are addressed by TGGE, which uses a temperature, rather than chemical, gradient to denature the sample.