In situ detection of genes in bacterial cells using flourescent dye-labeled polynucleotide probes (RING-FISH): Improvements of specificity and permeabilization as well as its application for environmental studies
Final Report Abstract
In situ hybridizations with polynucleotides require the adaption of per-meabilization procedures of the cells. In this project permeabilization procedures for the variety of cell walls/envelopes of diazotrophic and other organisms should be developed and optimized. Another major goal was the improvement of probe specificity of the RING-FISH procedure. The low specificity of RING-FISH should be overcome by the development and application of synthetic construct probes. A further aim was the evaluation of whether FISH using polynucleotides can be used for visualizing nifH gene expression at the cellular level and to combine this approach with conventional rRNA-targeting FISH. The presented studies in general illustrate the capacity of RING-FISH technology, but show also its current limitations concerning target accessibility and probe specificity. Individual adaptations of the protocols are needed for the respective target genes and organisms.
