Dgge

Denaturing gradient gel electrophoresis (DGGE) is based on the analytical separation of DNA fragments of identical or near-identical length based upon their sequence composition (78). Separation is based on the changing elec-trophoretic mobilities of DNA fragments migrating in a gel containing a linearly increasing gradient of DNA dénaturants (Fig. 6.4). Changes in fragment mobility are associated with partial melting of the double-stranded DNA in discrete regions, the so-called melting domains. Each band shown on the gel represents a taxonomic unit present in the environment, and the band intensity can be associated with the species' abundance within it. Once the gel has been visualized, it is also possible to directly cut out bands for sequencing.

The method derives from one used in the medical sciences, which was subsequently modified for microbial community analyses o

DNA fragment

Dénaturant conc. (formamide + urea)

High

DNA fragment

GC clamp

Dénaturant conc. (formamide + urea)

Dna Contaminated Bands

Mobility: low

Mobility: stop

FIGURE 6.4 DGGE principle, conc., concentration. After Iwamoto and Nasu (55).

GC clamp

Mobility: high

Mobility: low

Mobility: stop

FIGURE 6.4 DGGE principle, conc., concentration. After Iwamoto and Nasu (55).

(77), in which the procedure is performed on the total community nucleic acid. An example of a DGGE gel is shown in Fig. 6.5 (30). The communities shown were extracted from various points of a diesel-contaminated groundwater remediation system.

DGGE has been used successfully in many investigations of community structure. It is now one of the most widespread and well-established methods used to obtain culture-independent microbial profiles and is starting to be used in bioremediation studies. For example, the DGGE community profiles of shoreline plots containing buried oil showed that controls and plots amended with liquid fertilizer had similar patterns (96). However, DGGE revealed that the bacterial community in plots treated with oil and slow-release fertilizer changed rapidly, probably as a result of the higher concentrations of nutrients available in interstitial water. Whiteley and Bailey (127)

published evidence for highly structured bacterial communities within different compartments of a phenolic-remediating activated sludge plant. Fluorescent in situ hybridization (FISH) whole-cell targeting mirrored gross changes in community structure shown by DGGE. An issue in bioavailability during bioremediation has been addressed by using DGGE as a tool (42): different phenanthrene-utilizing bacteria inhabiting the same soils may be adapted for different phenanthrene bioavailabilities. The genus Burkholderia is itself diverse and is implicated regularly in bioremediation processes. DGGE analysis ofPCR. products has shown that there were sufficient differences in migration behavior to distinguish the majority of 14 Burkholderia species tested (99).

These sorts of studies reveal insights that either are impossible with traditional microbiological studies or else would involve much greater levels of effort, and the place of DGGE

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