Denaturing Gradient Gel Electrophoresis

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DGGE starts out like almost molecular phylogenetic analysis does these days; by the isolation of DNA from environmental samples, followed by PCR of ssu-rRNA genes. Rather than cloning and sequencing from this pool of genes, however, they are first separated into unique sequences based on their denaturation properties.

DGGE is carried out in polyacrylamide gels in which the concentration of urea and formamide increases from top to bottom in the gel; i.e. the gel contains a gradient of denaturants. (Remember that denaturation of DNA means separation of the two strands.) The PCR-amplified ssu-rDNA is loaded in wells at the top of the gel, where the concentration of urea/formamide is too low to denature the DNA. As the ssu-rDNA migrates down the gel during electrophoresis, the concentration of urea/formamide increases until, at some point, it is high enough to denature the DNA. At this point, the ssu-rDNA band essentially stops moving (it slows way down). Because every ssu-rDNA sequence will have a different denaturation point, they will denature at different levels of the gel and separate into distinct bands despite the fact that the ssu-rDNAs in all of the bands are all the same size.

DGGE

A technical improvement that has become standard in this method is the incorporation of a long tail of G=C basepairs to the end of one of the PCR primers. This "GC clamp" keeps the denatured strands of the ssu-rDNA from becoming completely separated, effectively doubling the length of the single-straded DNA (slowing it to a near stop), and so that the two strands don't begin to separate into two distinct bands in the gel, confusing the issue.

Another version of DGGE is TGGE; temperature gradient gel electrophoresis, in which the denaturant is temperature instead of urea/formamide. The electrophoresis unit is designed so that the gel is heated in a controllable fashion, usually to a higher temperature at the bottom than at the top.

The gels are stained once run and visualized as usual. Each visible band represents an abundant organism in that environment. The pattern of bands is a "fingerprint" of the microbial population of the environment. The intensity of each band represents the abundance of the organism, to some extent, and can be followed from place to place or time to time. In order to actually identify the organism represented by each band, you can cut the band from a gel, re-PCR amplify it, and sequence it.