ISSN:
1365-2958
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
,
Medicine
Notes:
Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 104−105 copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (101−103 copies per cell) and chromosomal DNA (〈10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method – a halo-like, ring-shaped concentration of fluorescence in the cell periphery – we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1046/j.1365-2958.2003.03834.x
Permalink
