Fluorescence itIn Situ Hybridization
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In situ hybridization describes the annealing of a labeled nucleic acid to complementary nucleic acid sequences in a fixed target (e.g., chromosomes, free nuclei, nuclei in tissue sections, and DNA) followed by visualisation of the location of the probe. Since its development about 30 yr ago (1,2), it has transformed into a highly effective and rapid technique for uses such as characterizing chromosome aberrations, gene mapping, and marker ordering as well as expression studies.
All in situ hybridization originally used radioactively labeled probes, and methodology for in situ hybridization using radioactive probes is covered in Chapter 13 by Poulsom. The strict regulations on radioactivity, long exposure times, and some practical difficulties with the use of radioactive labels limited the wide application of the technique. In the 1980s, several methods using non-radioactive labeling were developed (3-7). The ease and effectiveness of fluorescence methods (fluorescence in situ hybridization [FISH]) in particular have now almost rendered the radioisotopic techniques obsolete. FISH has been developed to incorporate chromosome painting and the analysis of the whole genome for aberrations using the approaches of comparative genomic hybridization (CGH; described in detail in Chapter 15 by Roylance), multifluor FISH (M-FISH), and spectral karyotyping (SKY).
In FISH, essentially the probes are labeled either directly or indirectly with various fluorochrome dyes such as fluorescein isothiocyanate (FITC) and tetramethyl rhodamine that fluoresce at different wavelengths when excited by ultraviolet (UV) light.
