Direct Sequencing of PCR-Amplified 23S rDNA
In prokaryotes, the ribosomal gene locus contains three rRNA species: 16S, 23S and 5S. The potential of sequencing 16S rDNA of bacteria and archaea for establishing phylogenetic relationships and for use in molecular diagnostics is well-documented. Currently, over 3000 16S rRNA sequences are available in the databases. However, the potential of the 23S rRNA gene as a diagnostic tool has not been fully explored. Strong secondary structure and a larger size (3 vs. 1.5 kb for 16S rDNA) have
... S rDNA) have hindered efficient sequencing of the 23S rRNA gene. So, there are just over 100 bacterial 23S rRNA sequences available for bacteria (4, 5, 8) . Commonly, 23S rRNA gene sequence is determined either by sequencing of cloned fragments (1) or by amplification of partial fragments and subsequent direct sequencing of these products (2, 9) . In a recent report on direct sequencing of the PCR-amplified 23S rDNA, Sallen et al. (9) described the preparation of the 23S rDNA by three amplification steps to obtain overlapping fragments and the subsequent use of 33 sequence primers to achieve the complete sequencing of the 23S rRNA gene. We describe a simple protocol that allows amplification of almost the complete 23S rRNA gene and the subsequent direct sequencing of the doublestranded fragment, thereby eliminating the need for cloning or preparation of single-stranded template (7). We adapted a sequencing strategy, developed by Amersham (Amersham International plc, Little Chalfont, Bucks, England, UK) for sequencing DNA clones, for direct sequencing of PCR-amplified 23S rDNA. We have chosen sequencing of the 23S rRNA gene of the human pathogen Francisella tularensisas a model to demonstrate the potential of the described strategy.