CiteULike: vrich's dgge

Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology

Gerard Muyzer — 2008-05-11T18:10:55-00:00

Antonie van Leeuwenhoek, Vol. 73, No. 1. (1 January 1998), pp. 127-141.

Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important.

Bacterial diversity of soils assessed by DGGE, T-RFLP and SSCP fingerprints of PCR-amplified 16S rRNA gene fragments: Do the different methods provide similar results?

Kornelia Smalla — 2008-05-05T17:08:35-00:00

Journal of Microbiological Methods, Vol. 69, No. 3. (June 2007), pp. 470-479.

Bacterial communities of four arable soils - pelosol, gley, para brown soil, and podsol brown soil - were analysed by fingerprinting of 16S rRNA gene fragments amplified from total DNA of four replicate samples for each soil type. Fingerprints were generated in parallel by denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism (SSCP) to test whether these commonly applied techniques are interchangeable. PCR amplicons could be separated with all three methods resulting in complex ribotype patterns. Although the fragments amplified comprised different variable regions and lengths, DGGE, T-RFLP and SSCP analyses led to similar findings: (a) a clustering of fingerprints which correlated with soil physico-chemical properties, (b) little variability between the four replicates of the same soil, (c) the patterns of the two brown soils were more similar to each other than to those of the other two soils, and (d) the fingerprints of the different soil types revealed significant differences in a permutation test, which was recently developed for this purpose.

Opening the black box of soil microbial diversity

James Tiedje — 2008-05-04T20:48:41-00:00

Applied Soil Ecology, Vol. 13, No. 2. (October 1999), pp. 109-122.

Soil probably harbours most of our planet's undiscovered biodiversity. Recent results from both, culturing and nucleic acid-based approaches indicate that soil microbial diversity is even higher than previously imagined. One reason for the high diversity is that much of the diversity can be found at very small scales. If the same genotypes are not repeated at other locations, the large-scale diversity is greatly multiplied. It remains to be seen to what extent this large genotypic diversity actually affects functional diversity, microbial ecology, or biotechnological significance. Here we present a framework of methods for opening the soil black box that provides different levels of resolution of both microbial community structure and activity. The rationale for and examples of use of three of these methods are presented: guanine plus cytosine content of total soil DNA (G+C), terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes amplified from soil DNA, and amplified ribosomal DNA restriction analysis (ARDRA) of rRNA genes from soil DNA and from isolates. These methods give coarse and moderate scale resolution of the soil community. The G+C method, which is one of the few comprehensive coarse scale methods, is also quantitative and can be used to separate DNA into G+C fractions for a second level of composition or activity analysis. The example of the ARDRA method used here illustrates that the same populations of 2,4-D degraders became dominant in three soils of very different land use history and that several of the 2,4-D degrading isolates from these sites had the same ARDRA pattern found from the soil DNA indicating that the isolates represent the dominant populations in the 2,4-D treated soil.

Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis

Markus Moeseneder — 2008-05-04T19:16:45-00:00

Appl. Environ. Microbiol., Vol. 65, No. 8. (1 August 1999), pp. 3518-3525.

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.