Amplification, Isolation and Direct Nucleotide Determination of Entire Genes: Application to the Study of 16S rRNAs for Molecular Evolution in Bacteria, Identification of Cultural Isolates and Development of Probes

Abstract

Molecular classification and identification of bacteria as well as the development of specific DNA probes is a tedious procedure often requiring multiple cloning steps. We demonstrate here a universal and general strategy which does not require any cloning steps suitable I) for inferring phylogenetic relationships, II) for characterization and definition of taxa at the molecular level and III) for the development of specific DNA probes. A target molecule fitted for such purposes should I) be present in all cellular organisms, II) consist of conserved and variable regions, III) contain a high information content and IV) be functionally constrained. Besides conserved proteins, e.g. cytochromes and heat-shock proteins, other conserved structures like ribosomal RNAs meet these conditions.