Overview of Fluorescence in Situ Hybridization Techniques For

Overview of Fluorescence In Situ UNIT 8.1 Hybridization Techniques for Molecular Cytogenetics HISTORY or immunocytochemistry): hence the term “in- In situ hybridization is a powerful technol- direct.” A number of such hapten modifications ogy for visualizing the location of specific have been described. Direct methods are also nucleic acid sequences on chromosomes, single amenable to immunocytochemical amplifica- cells, or tissue sections through the use of a tion if antibodies against the reporter molecules nucleic acid probe that is complementary to are available (Raap et al., 1990; Wiegant et al., those sequences and has been labeled in some 1991). fashion that renders it detectable. Until the early Haptens currently in use include biotin, di- 1980s, radioisotopes were the only labels avail- goxigenin, and fluorescein. Fluorescein tetra- able for nucleic acid probes and microautora- methyl rhodamine, aminomethyl coumarin ace- diography was the only means to detect in tic acid (AMCA), and a series of cyanin dyes situ–hybridized sequences. Radioactive probes are in widespread use as fluorochromes, provid- provide limited spatial resolution for in situ ing good spectral coverage across visual and hybridization because the decaying particles infrared wavelengths. Although chemical meth- leave tracks, not discrete spots, in the photo- ods for DNA labeling exist, generally haptens graphic emulsion. It is further limited by the and fluorochromes are incorporated enzymati- size of the silver halode crystals in the emul- cally into newly synthesized DNA using hapten- sion. Moreover, many practical inconveniences or fluorochrome-modified dUTP. The allyla- are imposed by the use of radioactivity, such as mine derivative of dUTP can be fluorochrom- the need to observe relatively complicated ized or haptenized, for example using N-hy- safety measures, the limited shelf life of radio- droxysuccinimide esters of haptens and fluoro- isotopes, and the long exposure periods re- chromes. Its use for enzymatic synthesis of quired by autoradiography. Finally, with radio- nonradioactive probes (Langer et al., 1981) was active detection it is not possible to distinguish a major achievement because it fit closely with multiple targets in one multiprobe in situ hy- existing molecular biology formats for the ra- bridization experiment. dioactive labeling of nucleic acids employing The development in the 1980s of stable DNA polymerases (e.g., by nick translation or nucleic acid labels that allowed nonradioactive random-primed labeling). This achievement detection through fluorescence or enzyme re- led to the widespread application of nonra- actions has demolished these practical and fun- dioactive probes in in situ hybridization. damental obstacles. In situ hybridization can Fluorescence in situ hybridization (FISH) now be performed rapidly with multiple differ- methods have achieved high standards of sen- ently colored nucleic acid probes at maximum sitivity, resolution, and multiplicity. In the fol- optical resolution, and this has permitted wide- lowing sections, these parameters and the con- spread application of this methodology in clini- ditions under which they are obtained are pre- cal and basic research. Because of the substan- sented, and molecular cytogenetic applications tial advantages offered by nonradioactive de- of FISH are briefly discussed. tection, the presentation of in situ hybridization techniques in this chapter is limited to those SENSITIVITY methods. The sensitivity of a FISH procedure is defined as the smallest nucleic acid sequence DIRECT VERSUS INDIRECT target detectable. In discussing FISH sensitiv- METHODS ity it is useful to consider a simplified view of In direct in situ hybridization, the fluores- the human genome as consisting of repeat and cent reporter molecule is bound to the nucleic unique sequences. Repeat sequences may be acid probe so that hybrids that have formed can clustered or dispersed. Relevant examples of be visualized microscopically immediately af- clustered repeats are alphoid DNAs, which are ter in situ hybridization (Wiegant et al., 1991). located at chromosome centromeres, and sim- In indirect procedures, the probe contains an ple satellite repeats, which occur at heterochro- element that renders it detectable by additional matic regions. Their clustered nature gives Molecular labeling steps (e.g., biotin-streptavidin binding them a high degree of chromosome specificity, Cytogenetics Contributed by A.K. Raap 8.1.1 Current Protocols in Cytometry (1997) 8.1.1-8.1.6 Copyright © 1997 by John Wiley & Sons, Inc. and their high copy number makes them readily feasible (Wiegant et al., 1996). Improvements detectable by FISH. Alu and Kpn repeats are in FISH sensitivity have recently been achieved examples of dispersely occurring repeats. Be- through the use of hapten- or fluorochrome-la- cause of their dispersed nature, these are not beled tyramides and horseradish peroxidase useful as markers and will increase background (Kerstens et al., 1995; Raap et al., 1995; van when present in probes. They are almost invari- Gijlswijk et al., 1996). This approach actually ably present in large-insert clones; therefore, an combines the sensitivity potentials of fluores- important technical issue when performing cence and enzyme-based detection schemes. It FISH with large genomic probes is the need to is of particular value in situations where the eliminate such dispersely occurring repeat se- signal-to-background ratio is suboptimal. quences from participation in the in situ hybridization reaction. This is done by preanneal- MULTIPLICITY ing the labeled DNA with unlabeled DNA en- The multiplicity of a FISH procedure is riched for repeats—i.e., C0t1 DNA—in a defined as the number of DNA targets that can process known as suppression in situ hybridi- be distinguished on the basis of optical proper- zation (Landegent et al., 1987; Lichter et al., ties, usually fluorescence color. In the simplest 1988; Pinkel et al., 1988). application of multiplicity, different fluoro- In metaphase chromosomes, unique targets chromes that are spectrally well separated are >30 to 40 kb (cosmid-insert size) are readily attached to separate probes either directly or detectable by microscopy after indirect FISH. indirectly. For the visible part of the electro- Generally, >90% of cells will show the ex- magnetic spectrum, blue, green, and red fluo- pected 2 × 2 copy number. Occurrence of paired rescent dyes are available, permitting a multi- spots on sister chromatids is a strong sign of plicity of 3 for visual observation of FISH specificity (Landegent et al., 1985; Lichter et results (Nederlof et al., 1989). When imaging al., 1990), and some nonspecific background devices sensitive to infrared light rays are used, spots may be tolerated. As probe size decreases, for example a CCD camera with light integra- detection efficiency drops to the point that at 1 tion capabilities, multiplicity can be increased to 2 kb of unique target, FISH detection effi- to 4 or 5. When the targets are spatially sepa- ciency is such that some statistical analysis is rated, as in well-spread metaphase chromo- necessary to assign the probe to a chromosomal somes, multiplicity can be increased by com- band. Successful sub-kb chromosomal FISH is binatorial labeling of the targets (Nederlof et rare. al., 1990; Ried et al., 1992a; Wiegant et al., In interphase cells, 30 to 40 kb of target is 1993; UNIT 8.3). Here multiplicity is 2n − 1, where also readily detectable by microscopy, but n is the number of spectrally resolvable fluoro- background should be reduced to a minimum chromes; this implies that with 3 and 4 fluoro- because—unlike in chromosomal FISH—no chromes, multiplicities of 7 and 15, respec- indicator of specificity is available. tively, can be achieved. For such combinatorial With DNA Fiber-FISH (Wiegant et al., labeling of multiple FISH targets the ratios of 1992), which uses naked DNA immobilized on the fluorescence intensities of the probes are in glass object slides, sensitivity is much better, principle not relevant, but because the fluores- most probably as a consequence of the high cence intensity ratios of differentially labeled accessibility of naked DNA to probes and im- probes recognizing the same target turn out to munological detection reagents. Genomic plas- be fairly constant after FISH (Nederlof et al., mids 1 to 2 kb in size are easily visualized 1992), multiplicity can easily be increased to (Florijn et al., 1995), although sensitivity is 12 (Dauwerse et al., 1992). Recently, success- better when hybridizing to larger genomic ful FISH imaging of all the human chromo- clones (e.g., cosmid). Recently, unique targets somes in 24 colors, using combinatorial FISH 200 bp in size have been detected, providing with five fluorochromes and either small band the means for (large) exon mapping (Florijn et excitation/emission filter sets or special spec- al., 1996). Digital imaging is recommended in tral imaging devices, has been reported such cases. (Schröck et al., 1996; Speicher et al., 1996). Indirect FISH methods are more sensitive Detection efficiency is an important issue in than direct ones (Wiegant et al., 1991). Direct multicolor FISH, and it is generally advisable methods are generally recommended for repeat to apply high-multiplicity FISH only to large targets such as satellite DNAs. Chromosomal targets. For example, with FISH detection effi- Overview of FISH for Molecular and interphase FISH using directly labeled cos- ciencies

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