Early Days
The broader historical setting for the development of cytochemical techniques in general is extensively and excellently reviewed elsewhere (van der Ploeg, 2000 ). We present a much-abridged history to describe the introduction, development and maturation of fluorescence in situ hybridization (FISH) specifically. In brief, the earliest histochemistry techniques consisted of the use of different sorts of natural and synthetic dyes to stain cellular structures and sub-cellular accumulations. These compounds were generally non-specific because they had affinities for certain general categories of molecules, be they basic proteins, nucleic acids, lipids or carbohydrates. Even the more specific stains for cellular accumulations and macromolecular complexes such as hemosiderin, amyloid, elastin and reticular fibers were not generally applicable to investigation of all the biomolecules of interest. The ability to detect specific molecular identities was first demonstrated using antigen-antibody interactions. Early in the 1940s, antibodies were conjugated to fluorochromes without loss of their epitope-binding specificity. Decades later, the first antibody-dependent fluorescent detection of nucleic acid hybrids was achieved (Rudkin and Stollar, 1977 ); however, this technology was soon replaced by the advent of fluorescent nucleic acid probes. The earliest in situ hybridizations, performed in the late 1960s, were not fluorescent at all, but rather utilized probes labeled with radioisotopes. Techniques not employing fluorescence, such as enzyme-based chromogenic reporters (reviewed by Hougaard et al., 1997 ) and gold-based probe systems used in electron microscopy (reviewed by Puvion-Dutilleul and Puvion, 1996 ) are each fields in their own right. Owing to space limitations, we cannot expand upon these topics further here; we focus our discussion specifically on FISH.
FISH for visualization of nucleic acids developed as an alternative to older methods that used radiolabeled probes (Gall and Pardue, 1969 ). Early methods of isotopic detection employed non-specific labeling strategies, such as the random incorporation of radioactive modified bases into growing cells, followed by autoradiography. Several drawbacks of isotopic hybridization inspired the development of new techniques. First, the very nature of radioactive material requires that the probe is unstable; the isotope decays over time and so the specific activity of the probe is not constant. Second, although sensitivity of radiography is generally high, resolution is limited. Third, long exposure times are often required to produce measurable signals on radiography film, delaying results of the assay. Fourth, radiolabeled probe is a relatively costly and hazardous material and it must be transported, handled, stored and disposed of in accordance with regulations. FISH allowed significant advances in resolution, speed and safety, and later paved the way for the development of simultaneous detection of multiple targets, quantitative analyses and live-cell imaging.
The first application of fluorescent in situ detection came in 1980, when RNA that was directly labeled on the 3' end with fluorophore was used as a probe for specific DNA sequences (Bauman et al., 1980 ). Enzymatic incorporation of fluorophore-modified bases throughout the length of the probe has been widely used for the preparation of fluorescent probes; one color is synthesized at a time (Wiegant et al., 1991 ). The use of amino-allyl modified bases (Langer et al., 1981 ), which could later be conjugated to any sort of hapten or fluorophore, was critical for the development of in situ technologies because it allowed production of an array of low-noise probes by simple chemistry. Low probe specific activity prevented the assessment of nucleic acids with low copy number by FISH. Methods of indirect detection allowed signal output to be increased artificially by the use of secondary reporters that bind to the hybridization probes. In the early 1980s, assays featuring nick-translated, biotinylated probes, and secondary detection by fluorescent streptavidin conjugates were used for detection of DNA (Manuelidis et al., 1982 ) and mRNA (Singer and Ward, 1982 ) targets. Approximately a decade later, improved labeling of synthetic, single-stranded DNA probes allowed the chemical preparation of hybridization probes carrying enough fluorescent molecules to allow direct detection (Kislauskis et al., 1993 ). Many variations on these themes of indirect and direct labeling have since been introduced, giving a wide spectrum of detection schemes from which to choose; the specifications, sensitivity and resolution of these techniques are well described elsewhere.
http://jcs.biologists.org/cgi/reprint/116/14/2833
Metaphase FISH
FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine Cytogenetics or identify extra material of unknown origin. It can also help in cases where it is difficult to determine from routine Cytogenetics if a chromosome has a simple deletion or is involved in a subtle or complex rearrangement. In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.
The number of microdeletion syndromes diagnosed by FISH is expanding rapidly. The sensitivity of these tests in every case is better than routine Cytogenetics but depends on the particular syndrome. In some syndromes, the probe is specific for the defective gene as in Williams Syndrome where a deletion has been shown in the elastin gene in 96% of individuals with a firm diagnosis. In other syndromes such as Prader-Willi/Angelman, the etiology of the syndrome is heterogeneous and microdeletions compose only a portion of the cases (60%).
http://www2.uah.es/biomodel/citogene/dynacare/fishinfo.htm
Interphase FISH
FISH can be used in interphase cells to determine the chromosome number of one or more chromosomes as well as to detect some specific chromosome rearrangements that are characteristic for certain cancers. The primary advantage of interphase FISH is that it can be performed very rapidly if necessary, usually within 24 hours, because cell growth is not required.
A good example is the Aneuploid Screen test which is performed on amniotic fluid cells when there is a strong clinical indication for one of the common trisomies. The sample nuclei are denatured and hybridized with DNA probes for chromosomes 13, 18, 21, X, and Y and results usually obtained within 24 hours. Routine Cytogenetics is included with an Aneuploid Screen to confirm the results or detect any abnormalities not detected by interphase FISH.
http://www2.uah.es/biomodel/citogene/dynacare/fishinfo.htm
Related Sites:
http://martin.parasitology.mcgill.ca/insituhybridization/INDEX.HTM
This site provides a good and clear overall description of In Situ Hybridization. It also has a cool slide show.
http://home.no.net/immuno/
This site provides detailed information about ISH, along with information about immunofluorescence and ISH procedures.
http://www.ksu.edu/wgrc/Protocols/Cytogenetics/ISH.html
This site gives several sample protocols for ISH.
http://www.findarticles.com/cf_dls/m2250/9_38/55812259/p1/article.jhtml
This site talks about FISH involved with clinical testing.
