In situ Hybridization
The technique of in situ hybridization is used to locate the chromosomal location of a specific DNA (or RNA) probe. The theory is the same as for Southern hybridization, except that the DNA to which the probe will hybridize is the actual chromosome. The probe is labeled with a flourescent probe. It is then added to a chromosomal preparation from the species of interest. After a sufficient time for annealing to occur, the chromosomes are viewed using a floruescent microscope. The probe will hybridize to the chromosome carrying the sequence of interest. If the species has been characterized cytogenetically, the marker can be assigned to the appropriate chromosome. Because this technique uses a flourescent probe it is called flourescence in situ hybridization or FISH. It has also be called chromosome painting.
Several additonal technical steps increaese the utility of the FISH technicque. If FISH is coupled with chromosome banding, the probe can be localized to a specific band of the chromosome. Chromosomes can also be characterized simulataneously with two probes, each labeled with a different flourescent label. This permits the ordering of any two probes on a specific chromosome. In this manner, physical linkages can be determined using FISH.
With an array of probes and chromosome banding, FISH analysis has been useful for studing human diseases. For example, if a patient suffering a disease is determined via FISH analysis to have a deletion at a specific chromosomal analysis, then the gene responsible is likely to reside on the missing segment. This experiment would involve analyzing the chromosome with a series of probes. If two specific probes do not hybridize as previous experiments predicted, then the candidate would reside on that deleted section. FISH analysis of tumor tissues revealed chromosomal additons and deletions may a characteristic of some cancers.
Finally, FISH revealed previously unknown features of nuclear topology. These analysis revealed that chromosomes do not reside in random positions in the nucleus, but actually seem to be located in specific locations. Further, the location of centromeres was not same for all cell types constant suggesting that the topological association with the nuclear cytoskeleton is not constant and may be under developmental conrol.Copyright © 1997. Phillip McClean