In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.
Obtaining information on the genetic capabilities and phylogenetic affinities of individual prokaryotic cells within natural communities is a high priority in the fields of microbial ecology, microbial biogeochemistry, and applied microbiology, among others. A method for prokaryotic in situ PCR (PI-PCR), a technique which will allow single cells within complex mixtures to be identified and characterized genetically, is presented here. The method involves amplification of specific nuclei acid sequences inside intact prokaryotic cells followed by color or fluorescence detection of the localized PCR product via bright-field or epifluorescence microscopy. Prokaryotic DNA and mRNA were both used successfully as targets for PI-PCR. We demonstrate the use of PI-PCR to identify nahA-positive cells in mixtures of bacterial isolates and in model marine bacterial communities.
Department of Marine Sciences, University of Georgia, Athens 30602-2206, USA.
R E
RE
Hodson
W A
WA
Dustman
R P
RP
Garg
M A
MA
Moran
D001483:Base Sequence; D002463:Cell Membrane Permeability; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D017753:Ecosystem; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008386:Marine Biology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011387:Prokaryotic Cells; D011550:Pseudomonas aeruginosa / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial