Molecular Cytogenetics
The introduction of fluorescent dyes into molecular biology techniques in the 1980s greatly improved techniques for visualizing DNA probes, and has allowed for the development of methods to visualize specific DNA sequences on human chromosomes using the microscope. The term "fluorescent in situ hybridization" (FISH) was adopted as a descriptive name for this methodology which combines techniques of molecular biology and cytogenetics (the study of chromosomes).
Using this technique, DNA probes are labeled with fluorescent dyes and hybridized to metaphase chromosome spreads on glass slides. DNA stains are used to visualize the unlabeled portions of the chromosomes. Thus, this method has become a means of identifying specific DNA sequences on metaphase chromosomes.
Initially, only single-color detection was possible, which limited most applications to the study of a single gene or region of the chromosome. The development of more sophisticated fluorescence filters and a variety of different dyes with their own unique fluorescence spectra, however, permit the simultaneous detection of multiple colored dyes, and thus, the analysis of multiple genes or chromosome regions in a single experiment. In addition, by using various combinations of five dyes, it is possible to discriminate 31 different dye combinations at a time.
There are a number of clever innovations in molecular cytogenetics, which have greatly increased the power of these methods beyond the simple applications of FISH. These include multiplex FISH (M-FISH) in which each chromosome is demarcated with a different color assigned by a computer software application based on the combination of fluorescent dyes present, spectral karyotyping (SKY) which produces a result similar to that in M-FISH using interferometry in place of standard fluorescence microscopy, cross-species color banding (RX-FISH) which uses probes produce from gibbons and other primates to produce a color banding pattern which is unique to each chromosome, padlock probes which interlock a circular fluorescent probe onto metaphase chromosomes potentially enabling detection of even subtle mutations which are not generally evident from chromosome studies, and primed in situ hybridization (PRINS) which uses the metaphase chromosome on a glass slide as a template for a PCR-type reaction to produce a result similar to that produced by conventional FISH methodology.
The introduction of each of these new tools to the molecular cytogenetic toolbox has increased the utility and flexibility of fluorescent microscopy in research and clinical applications in genetics. These techniques are used to identify genetic abnormalities in interphase or metaphase cells, to detect submicroscopic deletions and duplications associated with a growing variety of clinical syndromes, to accurately characterize complex chromosome rearrangements which occur frequently in tumor cells, and to compare the genomic organization of different species with each other.
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