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 are 0.9 and an attempted multiplicity Cytogenetics mids and YACs as probes is, however, also 8.1.2

Current Protocols in Cytometry of 6, only in 0.96 × 100, or 53%, of the cells Using total DNA from human (radiation) will all six targets be visible simultaneously. hybrid cell lines as probe, the chromosomal origin of the human component can be rapidly RESOLUTION mapped cytogenetically by FISH to normal FISH resolution is defined as the smallest metaphase chromosomes (Kievits et al., 1990). genomic distance that must exist between two Similarly, microdissected or flow-sorted ab- DNA sequences in order for them to be resolved normal chromosomes may be characterized by microscopically. Resolution is inversely pro- “reverse painting” following universal amplifi- portional to chromatin compaction and is lim- cation strategies (Carter et al., 1992; Melzer et ited by the spatial resolution of the microscope. al., 1993). Apart from the resolution limit, the range over Chromosomal imbalances in a test DNA which that resolution is available is a useful sample can be assessed and cytogenetically specification for probe ordering and mapping mapped using comparative genomic hybridiza- by FISH. For metaphase chromosomes, with tion (CGH). CGH is based on the simultaneous their highly condensed chromatin, maximum in situ hybridization of differentially labeled FISH resolution has been determined to be 1 to test DNA (e.g., tumor DNA; in red) and refer- 3 Mb, ranging up to the full genome (Lawrence ence DNA (e.g., normal DNA; in green) to et al., 1990; Lichter et al., 1990). FISH per- normal metaphase chromosome preparations. formed on more relaxed chromatin, such as that If over- or underrepresentation of DNA se- of interphase nuclei, provides a resolution of quences occurs in the test DNA, then an imbal- 100 kb up to 1 to 2 Mb (Lawrence et al., 1990; ance in the red-over-green fluorescence inten- Trask et al., 1991; van den Engh et al., 1992). sity ratio results at the cytogenetic location where FISH to naked DNA fibers provides the highest the over- or underrepresented DNA maps (Kal- resolution: 1 kb ranging up to 400 to 1000 kb lioniemi et al., 1992a; du Manoir et al., 1993). (Wiegant et al., 1992; Parra and Windle, 1993; Probes for whole chromosomes or parts Haaf and Ward, 1994; Heiskanen et al., 1994; thereof can be used to help characterize com- Senger et al., 1994; Florijn et al., 1995). With plex karyotypes; particularly when performed DNA Fiber-FISH, resolution is limited only by in high multiplicity, this considerably alleviates the spatial resolution of the light microscope; the problems associated with conventional kar- 1 kb of double-stranded DNA has a length of yotyping (Smit et al., 1991; Lengauer et al., 0.34 µm, which corresponds to the practical 1993; Schröck et al., 1996; Speicher et al., resolution limit of light microscopy (Florijn et 1996). al., 1995). Multicolor FISH using probes for chromosome-specific repeat targets is of great value in APPLICATIONS the detection of aneusomy (Hopman et al., With the specifications discussed above, 1988; Arnoldus et al., 1991; Ried et al., 1992b), FISH has great potential for DNA mapping as whereas cosmid, YAC, and especially BAC and well for analysis of the molecular genetic P1 clones provide excellent FISH probes for makeup of individual cells in a morphological targeted detection of regional deletions, ampli- context (van Ommen et al., 1995). As a conse- fications, and structural rearrangements (Ar- quence, the applications of FISH are manyfold, noldus et al., 1990; Tkachuk et al., 1990; Kal- and the technique has had a strong impact in lioniemi et al., 1992b; Ried et al., 1992c; Stil- clinical and research fields with a strong mor- genbauer et al., 1993; Bentz et al., 1994). phological component, such as cytogenetics, FISH also plays an essential role in cytoge- pathology, hematology, and cell biology, as netic and physical DNA mapping, because it well as in nonmorphological disciplines such provides a wide resolution range (see discus- as molecular genetics. FISH can be regarded as sion of Resolution for literature references). an elegant merger of molecular biology and Apart from these molecular (cyto)genetic cytology. applications, FISH has a role to play in molecu- In general, the ability of FISH to visualize lar cell biology because it permits detection of chromosomes and chromosome segments as gene expression at the RNA level (Lawrence et discrete domains is of prime importance, be- al., 1989; Dirks et al., 1993), investigation of cause it permits rapid assessment of the loca- the timing of replication (Kitsberg et al., 1993; tion, copy number, and rearrangements of Selig et al., 1993), and study of functional genes and chromosomes in individual cells, organization (Cremer et al., 1993; Zachar et al., irrespective of cell cycle stage (Cremer et al., 1993; Zirbel et al., 1993). Molecular 1986; Emanuel, 1993). Cytogenetics 8.1.3

Current Protocols in Cytometry LITERATURE CITED 1995. High-resolution DNA Fiber-FISH for Arnoldus, E.P.J., Wiegant, J., Noordermeer, I.A., genomic DNA mapping and colour-bar coding Wessels, J.W., Beverstock, G.C., Grosveld, G.C., large genes. Hum. Mol. Genet. 4:831-836. van der Ploeg, M., and Raap, A.K. 1990. Detec- Florijn, R.J., van de Rijke, F.M., Vrolijk, H., Blon- tion of the Philadelphia chromosome in inter- den, L.A.J., Hofker, M.H., den Dunnen, J.T., phase nuclei. Cytogenet. Cell Genet. 54:108- Tanke, H.J., van Ommen, G.J.B., and Raap, A.K. 111. 1996. Exon mapping by Fiber-FISH or LR-PCR. Arnoldus, E.P.J., Noordermeer, I.A., Peters, A.B.C., Genomics. In press. Voormolen, J.H.C., Bots, G.T.A.M., Raap, A.K., Haaf, T. and Ward, D.C. 1994. High resolution or- and van der Ploeg, M. 1991. Interphase cytoge- dering of YAC contigs using extended chromatin netics of brain tumors. Genes Chrom. Cancer and chromosomes. Hum. Mol. Genet. 3:629-633. 3:101-107. 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Multiple Kitsberg, D., Selig, S., Brandeis, M., Simon, I., colors by fluorescence in situ hybridization us- Keshet, I., Driscoll, D.J., Nicholls, R.D., and ing ratio-labelled DNA probes create a molecu- Cedar, H. 1993. Allele-specific replication timing lar karyotype. Hum. Mol. Genet. 1:593-598. of imprinted gene regions. Nature 364:459-463. Dirks, R.W., van de Rijke, F.M., Fujishita, S., van Landegent, J.E., Jansen in de Wal, N., van Ommen, der Ploeg, M., and Raap, A.K. 1993. Methodolo- G.J.B., Baas, F., de Vijlder, G., van Duijn, P., and gies for specific intron and exon localization in van der Ploeg, M. 1985. Chromosomal localiza- cultured cells by haptenized and fluoro- tion of a unique gene by non-autoradiographic in chromized probes. J. Cell Sci. 104:1187-1197. situ hybridization. Nature 317:175-177. du Manoir, S., Speicher, M.R., Joos, S., Schröck, E., Landegent, J.E., Jansen in de Wal, N., Dirks, R.W., Popp, S., Doehner, H., Kovacs, G., Robert-Ni- Baas, F., and van der Ploeg, M. 1987. Use of coud, M., Lichter, P., and Cremer, T. 1993. De- whole cosmid cloned genomic sequences for tection of complete and partial chromosome chromosomal localization by non-radioactive in gains and losses by comparative genomic in situ situ hybridization. Hum. Genet. 77:366-370. hybridization. Hum. Genet. 90:590-610. Langer, P.R., Waldrop, A.A., and Ward, D.C. 1981. Emanuel, B.S. 1993. The use of fluorescence in situ Enzymatic synthesis of biotin-labeled polynu- hybridization to identify human chromosomal cleotides: Novel nucleic acid affinity probes. anomalies. Growth Genet. Hormones 9:6-12. Proc. Natl. Acad. Sci. U.S.A. 78:6633-6637. Overview of FISH Florijn, R.J., Blonden, L.A.J., Vrolijk, H., Wiegant, Lawrence, J.B., Singer, R.H., and Marselle, L.M. for Molecular J., Vaandrager, J.W., Baas, F., den Dunnen, J.T., 1989. Highly localized tracks of specific tran- Cytogenetics Tanke, H.J., van Ommen, G.J.B., and Raap, A.K. 8.1.4

Current Protocols in Cytometry scripts within interphase nuclei visualized by in fluorochrome-tyramides. Hum. Mol. Genet. situ hybridization. Cell 57:493-502. 4:529-534. Lawrence, J.B., Singer, R.H., and McNeil, J.A. Ried, T., Baldini, A., Rand, T.C., and Ward, D.C. 1990. Interphase and metaphase resolution of 1992a. Simultaneous visualization of seven dif- different distances within the human dystrophin ferent DNA probes by in situ hybridization using gene. Science 249:928-932. combinatorial fluorescence and digital imaging Lengauer, C., Speicher, M.R., Popp, S., Jauch, A., microscopy. Proc. Natl. Acad. Sci. U.S.A. Taniwaki, M., Nagaraja, R., Riethman, H.C., 89:1388-1392. Donis-Keller, H., D’Urso, M., Schlessinger, D., Ried, T., Landes, G., Dackowski, W., Klinger, K., and Cremer, T. 1993. Chromosomal bar codes and Ward, D.C. 1992b. Multicolor fluorescence produced by multicolor fluorescence in situ hy- in situ hybridization for the simultaneous detec- bridization with multiple YAC clones and whole tion of probe sets for chromosomes 13, 18, 21, chromosome painting probes. Hum. Mol. Genet. X and Y in uncultured amniotic fluid cells. Hum. 2:505-512. Mol. Genet. 1:307-313. Lichter, P., Cremer, T., Borden, J., Manuelidis, L., Ried, T., Lengauer, C., Cremer, T., Wiegant, J., and Ward, D.C. 1988. Delineation of individual Raap, A.K., van der Ploeg, M., Groitl, P., and human chromosomes in metaphase and inter- Lipp, M. 1992c. Specific metaphase and interphase cells by in situ hybridization using recom- phase detection of the breakpoint region in 8q24 binant DNA libraries. Hum. Genet. 80:224-234. of Burkitt lymphoma cells by triple colour fluo- Lichter, P., Tang, C.C., Call, K., Hermanson, G., rescence in situ hybridization. Genes Chrom. Evans, G., Housman, D., and Ward, D.C. 1990. Cancer 4:69-74. High resolution mapping of human chromosome Schröck, E., du Manoir, S., Veldman, T., Schoell, B., 11 by in situ hybridization with cosmid probes. Wienberg, J.Y., Ferguson-Smith, M.A., Ning, Y., Science 247:64-69. Ledbetter, D.H., Bar-Am, I., Soenksen, D., Ga- Melzer, P.S., Guan, X.U., Burgers, A., and Trent, rini, Y., and Ried, T. 1996. Multicolor spectral J.M. 1993. Rapid generation of region specific karyotyping of human chromosomes. Science probes by chromosome microdissection and 273:494-497. their application. Nature Genet. 1:24-28. Selig, S., Okumura, K., Ward, D.C., and Cedar, H. Nederlof, P.M., Robinson, D., Abuknesha, R., 1993. Delineation of DNA replication time Wiegant, J., Hopman, A.H.N., Tanke, H.J., and zones by fluorescence in situ hybridization. Raap, A.K. 1989. Three-color fluorescence in EMBO J. 11:1217-1225. situ hybridization for the simultaneous detection Senger, G., Jones, T.A., Fidlerova, H., Sanseau, P., of multiple nucleic acid sequences. Cytometry Trowsdale, J., Duff, M., and Sheer, D. 1994. 10:20-27. Released chromatin: Linearized DNA for high Nederlof, P.M., Flier, S., Wiegant, J., Raap, A.K., resolution fluorescence in situ hybridization. Tanke, H.J., Ploem, J.S., and van der Ploeg, M. Hum. Mol. Genet. 3:1275-1280. 1990. Multiple fluorescence in situ hybridiza- Smit, V.T., Wessels, J.W., Mollevanger, P., tion. Cytometry 11:126-131. Dauwerse, J.G., van Vliet, M., Beverstock, G.C., Nederlof, P.M., Flier, S., Vrolijk, J., Tanke, H.J., and Breuning, M.H., Devilee, P., Raap, A.K., and Raap, A.K. 1992. Fluorescence ratio measure- Cornelisse, C.J. 1991. Improved interpretation ments of double-labeled probes for multiple in of complex chromosomal rearragements by situ hybridization by digital imaging micros- combined GTG banding and in situ suppression copy. Cytometry 13:839-845. hybridization using chromosome-specific libraries and cosmid probes. Genes Chrom. Cancer Parra, I. and Windle, B. 1993. High resolution visual 3:239-248. mapping of stretched DNA by fluorescent hybridization. Nature Genet. 5:17-21. Speicher, M.R., Gwyn Ballard, S., and Ward, D.C. 1996. Karyotyping human chromosomes by Pinkel, D., Landegent, J.E., Collins, C., Fuscoe, J., combinatorial multi-fluor FISH. Nature Genet. Segraves, R., Lucas, J., and Gray, J. 1988. Fluo- 12:368-375. rescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy Stilgenbauer, S., Dohner, H., Bulgay-Morschel, M. 21 and translocations of chromosome 4. Proc. Weitz, S., Bentz, M., and Lichter, P. 1993. High Natl. Acad. Sci. U.S.A.85:9138-9142. frequency of monoallelic retinoblastoma gene deletion in B-cell chronic lymphocytic leukemia Raap, A.K., Nederlof, P.M., Dirks, R.W., Wiegant, shown by interphase cytogenetics. Blood J.C.A.G., and van der Ploeg, M. 1990. Use of 81:2118-2222. haptenised nucleic acid probes in fluorescent in situ hybridisation. In In Situ Hybridization: Ap- Tkachuk, D.C., Westbrook, C.A., Andreeff, M., plication to Developmental Biology and Medi- Donlon, T.A., Cleary, M.L., Suryanarayan, K., cine (N. Harris and D.G. Williams, eds.) pp. Homge, M., Redner, A., Gray, J., and Binkel, D. 33-41. Cambridge University Press, Cambridge. 1990. Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization. Raap, A.K., van de Corput, M.P.C., Vervenne, Science 250:559-561. R.A.W., van Gijlswijk, R.P.M., Tanke, H.J., and Wiegant, J. 1995. Ultrasensitive FISH using per- Trask, B.J., Massa, H., Kenwrick, S., and Gitschier, Molecular oxidase-mediated deposition of biotin- or J. 1991. Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization Cytogenetics 8.1.5

Current Protocols in Cytometry of DNA sequences in interphase cell nuclei. Am. Rudkin, G.T. and Stollar, B.D. 1977. High resolu- J. Hum. Genet. 48:1-15. tion detection of DNA⋅RNA hybrids in situ by van den Engh, G., Sachs, R., and Trask, B.J. 1992. indirect immunofluorescence. Nature 265:472- Estimating genomic distance from DNA se- 473. quence location in cell nuclei by a random walk First description of indirect FISH using anti- model. Science 257:1410-1412. DNA⋅RNA antibodies. van Gijlswijk, R.P.M., Wiegant, J., Raap, A.K., and Bauman, J.G.J., Wiegant, J., Borst, P., and Van Tanke, H.J. 1996. Improved localization of fluo- Duijn, P. 1980. A new method for fluorescence rescent tyramides for fluorescence in situ hy- microscopical localization of specific DNA se- bridization using dextran sulfate and polyvinyl quences by in situ hybridization of fluorochrome alcohol. J. Histochem. Cytochem. 44:389-392. labeled RNA. Exp. Cell Res. 138:485-490. van Ommen, G.J.B., Breuning, M.H., and Raap, First report of direct FISH using fluorochrome-la- A.K. 1995. FISH in genome research and mo- beled RNA. lecular diagnostics. Curr. Opin. Genet. Devel. 5:304-308. Langer et al., 1981. See above Wiegant, J., Kalle, W., Mullenders, L., Brookes, S., First description of the synthesis of allylamine Hoovers, J.M.N., Dauwerse, J.G., van Ommen, dUTP, its conjugation to biotin, and its use in DNA G.J.B., and Raap, A.K. 1992. High-resolution in polymerase reactions. situ hybridization using DNA halo preparations. Hum. Mol. Genet. 1:587-591. Landegent et al., 1985. See above. Wiegant, J., Ried, T., Nederlof, P.M., van der Ploeg, First report of single-copy gene detection by nonra- M., Tanke, H.J., and Raap, A.K. 1991. In situ dioactive in situ hybridization using reflection mi- hybridization with fluoresceinated DNA. Nucl. croscopy. Acids Res. 19:3237-3241. Cremer et al., 1986. See above. Wiegant, J., Wiesmeijer, C.C., Hoovers, J.M.N., Schuuring, E., d’Azzo, A., Vrolijk, J., Tanke, Introduction of “interphase cytogenetics.” H.J., and Raap, A.K. 1993. Multiple and sensitive fluorescence in situ hybridization with rho- Landegent et al., 1987; Lichter et al., 1988; and damine-, fluorescein-, and coumarin-labeled Pinkel et al., 1988. See above. DNAs. Cytogenet. Cell Genet. 63:73-76. First reports of the repeat suppression hybridization Wiegant, J., Verwoerd, N.P., Mascheretti, S., Bolk, principle, also called chromosomal in situ suppres- M., Tanke, H.J., and Raap, A.K. 1996. An evalu- sion hybridization. ation of a new series of fluorescent dUTPs for Wiegant et al., 1992; Parra and Windle, 1993. See fluorescence in situ hybridization. J. Histochem. above. Cytochem. 44:525-529. First Fiber-FISH reports. Zachar, Z., Kramer, J., Mims, I.P., and Bingham, P.M. 1993. Evidence for channeled diffusion of Hopman et al., 1986. See above. pre-mRNA during nuclear RNA transport in metazoans. J. Cell Biol. 12:729-742. First bicolor FISH paper. Zirbel, R.M., Mathieu, U.R., Kurz, A., Cremer, T., Kallioniemi et al., 1992a. See above. and Lichter, P. 1993. Evidence for a nuclear First CGH report. compartment of transcription and splicing located at chromosome boundaries. Chromosome Res. 1:93-106. Contributed by A.K. Raap KEY REFERENCES Leiden University Pardue, M.L., and Gall, J.G. 1969. Molecular hy- Leiden, The Netherlands bridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl. Acad. Sci. U.S.A. 64:600-604. Very first report of in situ hybridization using autoradiography for detection.

Overview of FISH for Molecular Cytogenetics 8.1.6

Current Protocols in Cytometry