Reverse Transcriptase Sequencing of Ribosomal RNA for Phylogenetic Analysis

This chapter focuses on reverse transcriptase sequencing of ribosomal RNA for phylogenetic analysis. The cyanobacteria comprise a large group of structurally complex and ecologically important gram-negative prokaryotes whose taxonomic treatment has generated much interest and controversy over the years. By molecular taxonomic criteria (particularly 16 S rRNA oligonucleotide catalog analysis), the cyanobacteria, together with chloroplasts and prochlorophytes, are a coherent, eubacterial grouping that warrants phylum status. The sequencing protocol is a variation of the base-specific, dideoxynucleotide-terminated, chain-elongation method. It has been modified for the use of reverse transcriptase and ribosomal RNA templates. The method relies on the fact that certain regions of 16 S rRNA nucleotide sequence vary little or not at all among different organisms. A short oligodeoxynucleotide, complementary to such a universally conserved site, specifically anneals to its target site on the 16 S rRNA, even in the presence of other cellular RNAs. The RNA purification method is designed for use on pure cultures containing only one type of 16 S rRNA. Mixed cultures, however, can often be sufficiently purified by differential centrifugation or filtration, at the time of harvesting, for the method to work.