Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells

Abstract

Examination of collections of 16S rRNA sequences revealed sequence domains that were unique to (and invariant within) the three primary lines of cellular descent: the archaebacteria, the eubacteria, and the eucaryotes. Oligodeoxynucleotides complementary to these conserved sequence domains were synthesized and used as hybridization probes. Each of the radiolabeled probes specifically hybridized to nylon membrane-bound 16S rRNA from the targeted kingdom. A probe complementary to a universally conserved sequence in 16S rRNAs was used as a positive control, while its complement provided a negative control for nonspecific binding. The abilities of the probes to bind specifically to whole, fixed cells representing a broad array of phylogenetic diversity were tested in whole-cell dot blot assays. Again, all of the probes specifically bound the targeted groups. By microautoradiography, the method was extended to permit phylogenetic identification of single cells microscopically.