The 16S rRNA is a ribonucleic acid molecule found in the ribosome. It has a structural role in the ribosome and a functional role in protein synthesis. The sequence of the rRNA molecule is highly conserved between organisms but it has regions that change more rapidly than others. These properties make the rRNA molecule and its gene sequence a useful tool for comparing relatedness between two or more organisms. So, the 16S rRNA ribosomal RNA gene sequences of bacteria and achaea have been the primary tools for identifying the populations inhabitating soil and for monitoring microbial community dynamics.

The 16S rRNA gene has been the basis of defining microbial community diversity for several decades. Functional genes or the intergenic spacer region are sequenced or used for denaturing gradient gel electrophoresis (DGGE) or terminal restriction fragment length polymorphism (T-RFLP), which are used to study community structure between treatments, environments or to confirm 16S rRNA based phylogeny.

Soil is one of the most diverse habitats on Earth. No two soils are the same. Bacteria play a central role in the rhizosphere, which is a complex and dynamic environment that varies temporally, spatially, and with different agricultural practices that are likely to influence the bacterial community.

Molecular analysis of bacterial diversity in terrestrial ecosystems, most frequently involves retrieval of 16S rRNA gene sequences by PCR amplification of extracted and purified nucleic acids, using broad-range or group-specific primer sets, along with subsequent analysis by cloning and characterization of clones by sequencing or T-RFLP.. Alternatively, fingerprinting of total PCR products may be carried out by using, for example, amplified ribosomal DNA (rDNA) restriction analysis, length heterogeneity PCR, single-strand conformation polymorphism and T-RFLP. The most frequently used community fingerprinting methods are denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis, which separate sequences on the basis of differences in denaturing properties, and hence migration distances, in chemical and temperature gradients, respectively. Fingerprinting methods allow more rapid comparison of samples and are generally used to detect shifts in populations over time and/or under different environmental conditions.

The 16S rRNA gene has remained tool for environment microbiology due to the enormous number of 16S rRNA gene sequence in the Ribosomal Database Project (RDP). The sequences from organisms isolated from soil or found is soil clone libraries are poorly related to any previously sequenced organisms. Comparing sequences to the available databases is a useful first step toward characterizing an unknown sequence. Full-length 16S rRNA sequences with >97% are considered to be from the same species. Identification of microbial species based on 16S rRNA should be corroborated with genetic or phenotypic evidence. Careful alignment of the unknown sequences to a collection (GenBank or The Ribosomal Database Project) of similar and dissimilar sequences. The aligned sequences can be compared using algorithms to determine their relatedness and to construct a phylogenetic tree. This phylogenic tree helps top find out their divergence from a common ancestor.

The cloning approach has provided lists of sequence percentages or restriction fragment length polymorphism classes, along with their relative amounts in libraries. Quantification of data recovered in rDNA libraries is limited by the restricted number of clones that can feasibly be screened, but data have been used to calculate indices of diversity. Clone libraries of the 16S rRNA gne can be used to estimate community composition and diversity. A clone library contains a collection of 16S rRNA gene from the amplified polymerase chain reaction (PCR) product for a sample. The frequency of sequences within a clone library will reflect the frequency of the sequence of the organism. Clones can be grouped by restriction fragments pattern, known as operational taxon units (OTUs). The representative of each OTU can be sequences and the frequency of unique OTUs can be counted and analysed with ecological measures. New statistically tools for analysing DNA clone libraries directly have been developed that assign clone sequences from a microbial community into OTUs based on their sequences. This can be used to provide an estimation of species richness and diversity.

Finally, it is concluded that the 16S rRNA gene is the preferred gene target for identification of bacterial species, defining microbial community diversity and establishing phylogenetic relationship between unknown and uncultivated soil microorganisms.

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Ph.D. Molecular Biology and Biotechnology

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