Recovery and Analysis of Ribosomal RNA Sequences from the Environment

Since the 1980s the use of ribosomal RNA (rRNA) sequence-based analysis to characterize microbial populations (mainly bacterial and archaeal populations) has increased significantly. This increased use is in response to the recognition that culture-based methods grossly misrepresent the composition of microbial populations as they occur in nature (1 ). To circumvent the biases inherent in culture-dependent studies of microbial communities, it was suggested that, by extraction of nucleic acids directly from environmental samples, genes that were present in all taxa could be isolated and sequenced (2 ,3 ). Comparative analysis of sequences recovered from environmental samples with those from cultured isolates would permit phylogenetic relationships of the uncultured taxa to be determined (2 ,3 ). The universally distributed genes most commonly used for such analyses are the rRNA genes, particularly those encoding the small ribosomal subunit RNAs (16S and 18S rRNA). rRNA genes have many advantages over other candidate genes, including the following:

1.	They are crucial components of ribosomes.
2.	They possess a common, essential function in all cells.
3.	Functional necessity constrains their primary and secondary structure and hence the degree of divergence in different taxa.
4.	Their primary structure is a mosaic of conserved and variable tracts of sequence. This permits unambiguous alignment of homologous positions in an rRNA sequence and identification of universally conserved and taxon-specific sequence motifs.
5.	There is little evidence of horizontal transfer of rRNA genes.
6.	Extensive rRNA reference sequence databases exist.
7.	A “tree of life” based on rRNA sequences provides a framework within which sequences recovered from natural samples can be accommodated.