Fluorescence In Situ Hybridization (FISH)

Fluorescence In Situ Hybridization (FISH)

FISH is a technique used to identify and localize the presence or absence of specific DNA sequences on cells and tissues.

Principles
The technique can detect target molecules (sequences of DNA or RNA) in single cells.   In general, nucleic acid probes are used to hybridize to whole cell chromosomes. If a given cell contains that gene of interest, the probe will hybridize to its complementary DNA.   If the probe has a fluorescent tag, it can be visualized under a fluorescent microscope.

Procedure

1. Fix the cells to a slide and make their cell envelopes permeable.

2. Add a selected “probe.” A probe is a short molecule of ss-DNA (an oligo-nucleotide) with a fluorescent molecule attached.   Probes can be general (that is, common to a large group of organisms like all Eubacteria) or specific (that is, common to a specific species or strain of species).   Probes made to 16s rRNA sequences can be used to identify that types of bacteria seen.   Probes could also be made to genes of specific proteins, to determine if cells can carry out certain functions (e.g. break down certain organic compounds).

3. Rinse unattached probe off.

4. View under microscope.

Advantages

• Could observe and identify individual cells in natural samples

• Could use multiple probes to investigate complex systems

• Probes could be general or very specific

Disadvantages

• Difficult for probes to permeate some cells

• Must carefully choose probes so that there is no cross reactivity

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Fluorescence in Situ Hybridization