In Situ Hybridization

To achieve sufficient sensitivity for detection, a large DNA probe is necessary.
The use of large DNA probes is complicated by the interspersion of highly repeated DNA among gene sequences in many species.
The use of unlabeled repeated DNA as competitor effectively eliminates the non- specific background.
Both sister chromatids should have fluorescent dots at the same position to allow a confident assignment of a molecular probe to a band on a chromosome.
Since a variety of labeling groups and a variety of fluorescent groups can be used, several markers can be mapped at the same time.
Hybridization of small probes (eg. 1kbp) without competitor DNA to polytene chromosomes should be effective in locating sequences on these chromosomes due to their higher copy number.

The use of prometaphase chromosomes (in the process of condensing) improves the resolution of the method.
Gene deletions can be detected via FISH images.
Advances in microscopy allow the simultaneous analysis of multiple probes. This provides a way of staining maize chromosomes to substitute for G- or Q- banding which does not work well with maize chromosomes.
Examine an image of FISH stained chromosomes and of painted chromosomes.
Chromosome painting has clinical applications.

E-mail inquiries to U. Melcher------------Last Updated: 15 August, 2009

		Metaphase chromosome squashes are prepared for hybridization. The probe for hybridization has been tagged with moieties (biotin, digoxigenin, dinitrophenol) that can be recognized by specific proteins (avidin, antibodies). After hybridization, the binding protein conjugated with a fluorescent moiety (fluorescein, rhodamine, Texas red, etc.) is added and allowed to bind specifically.

At right are diagrammed the results of three different hybridization regimes: a 1kbp cloned probe containing an exon of a single copy gene (left); a 40kbp probe encompassing A (middle); the same probe, but with an excess of unlabeled highly repetitive DNA from the target species (right). The technique is called fluorescent in situ hybridization (FISH).