DIRECT RIBOSOMAL-RNA SEQUENCING FOR PHYLOGENETIC STUDIES

This chapter discusses direct ribosomal RNA sequencing for phylogenetic studies. Primer extension with reverse transcriptase in the presence of chain-terminator dideoxynucleotides is a straightforward approach for direct RNA sequencing. A variation of this method largely extends its usefulness and range of application, particularly for systematic studies in the field of molecular evolution. Selection of the primer sequence within a highly conserved region of the RNA allows for direct RNA sequencing on numerous organisms with the same primer. This sequencing method can be applied to any molecular species of RNA, provided a strongly conserved region can be identified in its sequence. Ribosomal RNAs (rRNAs) constitute choice targets for this sequencing approach. Their high relative abundance in cellular RNA permits direct determinations on total unfractionated RNA and their complex mosaic pattern of variation provides the basis for refined evaluations of phylogenetic distances. Universal primers for rRNA sequencing can be selected within a set of almost perfectly conserved sequence motifs scattered along both the small and large subunit molecules. © 1993