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Fish Technique: A Powerful Technique For The In Situ Detection Of Organisms In Environmental Samples

578 words - 3 pages

FISH is a powerful set of technique for the in situ detection of active growing organisms in environmental samples, a procedure that allows researchers to locate the positions of particular DNA or RNA sequences in the cytoplasm, organelles or nucleus [59]. In FISH a labeled DNA or RNA sequence is used as a probe to identify or quantify the natural occurring counterpart of the sequence in a biological sample. FISH probes often target sequences of rRNA or mitochondrial genes because they are abundant in sequence databases and in multiple copies in each cell. An evolution of classical oligonucleotide probes are peptide nucleic acid (PNA) probes, which are synthetic oligomers mimicking the DNA or the RNA structure. In PNA probes, the negatively charged (deoxy) ribose-phosphate nucleic acid backbone is replaced by an uncharged dN-(2-aminoethyl)-glycine scaffold to which the nucleotide bases are attached via a ...view middle of the document...

Limitations of the FISH method can include fungal and substrate inherent autofluorescence, insufficient permeability of cell walls, non-specific binding of probes, and low ribosome contents. Autofluorescence of fungi can also lead to fals epositive fluorescence signals. It is, therefore, important to check for autofluorescence of both target and non-target organisms before selecting the fluorochrome for probe labelling. However, the pin-hole technique of a laser scanning microscope is helpful in reducing background emissions compared to conventional epifluorescence. Low or no permeability of fungal cell walls can lead to weak or no signals because of the failure of FISH probes to penetrate rigid cell walls. One possible way to overcome this problem is to use cell wall lysing enzymes, such as chitinases and glucanases. A more elegant method is the use of peptide nucleic acid (PNA) probes. PNA probes are mimics in which the negatively charged sugar-backbone of DNA is replaced with a non-charged polyamide backbone. PNA probes share the same nucleotide bases as theconventional DNA probes. However, PNA probes penetrate cell walls more effectively due to their neutrality and they do not have to overcome the destabilizing electrostatic repulsion during hybridization. Low ribosome contents can arise from either a scarcity of potential substrate in oligotrophic habitats, or the presence of components inhibiting fungal growth. However, the Catalysed Reporter Deposition FISH (CARD-FISH) is a variant of the FISH method designed to enhance probe conferred signals. The major difference is that the probes are labelled with horse-radish-oxidase instead of a fluorochrome. Fluorescence labeled tyramide is then added to the cells after hybridizations. The horse-radish-oxidase catalyses the deposition of the reporter and this reaction leads to 20 to 30 fold stronger FISH signals than conventional FISH. Consequently, the number of active rRNA molecules necessary to detect a probe conferred signal decreases by using CARD FISH.

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