Fluorescent in situ Hybridization (FISH)

This page briefly presents the fluorescent in situ hybridization technique (FISH). This technique is used for the detection of target molecules with a system of coupled antibodies and fluorochromes. More details can be found in the cited references and links of the References section.

The detection of nucleotidic sequences on a combed DNA molecule is peformed indirectly, by first hybridizing the seeked nucleotidic sequences (the probes) with the combed DNA (also called the matrix DNA or target). If the probes are synthesized with incorporated fluorescent molecules or antigenic sites which can be recognized with fluorescent antibodies, the direct visualisation of the relative position of the probes is possible. This is the goal of physical mapping.

The probes are synthesized separately using different existing protocols. Basically, one resynthesized one strand of a double strand DNA molecule while incorporating modified nucleotides.

The random priming technique which we mostly use, consist in the polymerisation of part of a complementary single strand between two single strand hexamers (short single strand DNA made of 6 nucleotides). This is explained on the following figure:

The polymerase enzyme P synthesized the complementary strand N between hexamer 1 and hexamer 2, previously hybridized onto the single strand DNA molecule M. During the synthesis, random incorporation of labelled nucleotides (the pink dots) takes place, which leads to a labelled single strand DNA molecule after denaturation.

The hybridization step consists in simply mixing the single strand probes with the denaturated target DNA (the combed molecules). Denaturation of the DNA is obtained by heating the DNA, which separates the two strands, and allows access of the single strand probes to their respective complementary combed single strand.

The detection of the probes is the final step of fluorescent in situ hybridization. It consists in recognising the probes with fluorescent antibodies corresponding to the antigenes incorporated in the probes (see above). In the biotin-avidin system, one uses modified fluorescent avidin molecules, which can themselves be recognized by another layer, as shown on the following figure:

M represents the denaturated DNA matrix strand, and H the hybridized probe. The probe has modified nucleotidic sites, which possess a biotin molecule, to which fluorescent streptavidin molecules spontaneously bind. These molecules are then recognized by anti-avidin antibodieswith a fluorescent site and a biotin arm. The same construction can be used several times, leading to a sandwich of detection layers.

Observation of the hybridized sequences is made with an epifluorescence microscope. The white light of the source lamp is filtered so that only the relevant wavelengths for excitation of the fluorescent molecules arrive onto the sample. Emission of the fluorochromes happens in general at larger wavelengths, which allows to distinguish between excitation and emission light by mean of another optical filter. One thus sees bright colored signals onto a dark background.

With a more sophisticated filter set, it is possible to distinguish between several excitation and emission bands, and thus between several fluorochroms, which allows observation of many different probes on the same strand.

An example of a bicolor hybridization can be seen in the physical mapping page.

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© DNA Biophysics Laboratory, Pasteur Institute	page designed by Xavier Michalet last revised: december 10th,1996