Microbial community analysis: Conventional methods

It is generally known among microbiologists that there is a huge potential of prokaryotic diversity made up of hitherto uncultured micro-organisms (Pace, 1997; Ward et al., 1990). Molecular techniques directed toward analysing the community composition of environmental samples indicate that hitherto classified prokaryotic species account for only the tip of the iceberg, considering the huge number (estimated as 4-6 x 1030) of undiscovered prokaryotes present on Earth (Whitman et al., 1998). Usually, profiles of microbial communities in environments have been surveyed using genetic fingerprinting methods. Genetic fingerprinting is a DNA-based technique which generates a fingerprint, the barcode-like DNA fragment pattern. This is a direct analysis of whole genomes extracted from environments or PCR products of selected genes amplified from environmental DNA, based on either sequence polymorphism or length polymorphism. These techniques include denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), terminal restriction fragment length polymorphism analysis (T-RFLP), single-strand conformation polymorphism (SSCP), random amplified polymorphic DNA (RAPD), ribosomal intergenic spacer analysis (RISA), length heterogeneity PCR (LH-PCR), amplified ribosomal DNA restriction analysis (ARDRA) and DNA microarrays. In general, genetic fingerprinting techniques are simple and rapid, and allow simultaneous analyses of a large number of multiple samples. The “fingerprints” from different samples are then compared using computer-assisted cluster analysis and community relationships or differences between microbial communities are inferred (Rastogi and Sani, 2011). However, fingerprinting approaches do not provide direct taxonomic identities of the members comprising the microbial community. Building up a comparable database is also impossible for fingerprinting-based methodology due to the variability of fingerprinting patterns depending on the gel-electrophoresis conditions.

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