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FISH (Fluorescent in situ hybridization) is a cytogenetic technique which can be used to detect and localize DNA sequences on chromosomes. It uses fluorescent probes which bind only to those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome.
Process
First, a probe is constructed. The probe has to be long enough to hybridize specifically to its target (and not to similar sequences in the genome), but not too large to impede the hybridization process, and it should be tagged directly with fluorophores, with targets for antibodies or with biotin. This can be done in various ways, for example nick translation and PCR using tagged nucleotides.
Then, a chromosome preparation is produced. The chromosomes are firmly attached to a substrate, usually glass. After preparation the probe is applied to the chromosome DNA and starts to hybridize. In several wash steps all unhybridized or partially hybridized probes are washed away. If signal amplification is necessary to exceed the detection threshold of the microscope (which depends on many factors such as probe labelling efficiency, the kind of probe and the fluorescent dye), fluorescent tagged antibodies or streptavidin are bound to the tag molecules, thus amplifying the fluorescence.
Finally, the sample is embedded in an anti-bleaching agent and observed on a fluorescence microscope.
Interphase FISH
In interphase FISH the probe is applied to preparations with intact nuclei, either cytospins, paraffin sections, or even nuclei extracted from paraffin blocks. Hybridization is carried out similarly. The fluorescent signals are seen as dots in the cell nucleus, which is usually counterstained with a dye that recognizes DNA.Applications
FISH can be used to map sequences to a specific position on a chromosome. While there are other ways to do this, the main advantage of FISH is that it is not dependent on recombination and thus can be used in chromosome regions where recombination is suppressed, such as the centromere region. It can be used to map repetitive sequences that occur at several places on a chromosome.
FISH can also be used to do chromosome painting to make a comparison between two species or varieties by using DNA from entire chromosomes or even the entire genome of one species/variety as a probe on the other. In this way, Chromosomal abnormalities can be identified and evolutionary relations can be deduced.
Finally, FISH can be used to identify microorganisms.
External links
- Immunohistochemistry - In Situ Hybridization
- Atlas of Genetics and Cytogenetics in Oncology and Haematology
"Fluorescent in situ hybridization"