In Situ Hybridization

Forming the hybrid is not sufficient to allow it to be mapped, because the molecules are too small to be seen in the microscope. Thus, to map the geographical distribution of the gene or gene product, the cytochemist applies sensitive detection systems that allow the hybrid to be seen. This is done by labeling the cDNA or cRNA probe itself with a molecule that can either be visualized directly (such as fluorescein, which glows when exposed to fluorescent light in a microscope) or indirectly (such as radioactive sulfur, which can be detected by autoradiography; or biotin, which can be detected by avidin or by specific antibodies to biotin). Whichever type of molecule is chosen, it should be small, so that it does not interfere with the hybridization process.

The molecule used to visualize the hybrid is called the reporter molecule because it "reports" the site of the hybridization of the probe to the cellular DNA or RNA. Many different detection systems are available to cytochemists, employing a wide variety of reporter molecules. These include fluorescent compounds, colloidal gold compounds, or enzyme reactions or radioactive elements. Cytochemists will choose the most sensitive detection system that is also appropriate for their laboratories. For example, laboratories that do not want to work with radioactive compounds may choose one of the many nonradioactive methods.

One of the best-known nonradioactive in situ hybridization methods is FISH or "fluorescence in situ hybridization." It allows the detection of many genes in a chromosome or a nucleus, and different combinations of fluorescent reporter molecules are used to produce different colors. Using FISH, a veritable rainbow of colors can be used to map the location of genes on chromosomes. Another method uses enzyme reactions to form a product over the site of the mRNA or DNA in the cell. One can use different enzymes or enzyme substrates in the detection system and thus detect multiple gene products in the same tissue or cells.