~
ECHNICAL ~'~ OCUS
F l u o r e s c e n t molecules can be deposited in chromatin at the sites of specific DNA sequences by use of fluorescence in situ hybridization (FISH). The technique is a simple one. DNA or RNA sequences are first labeled with reporter molecules. The probe and the target chromosomes or nuclei are denatured. Complementary sequences in the probe and target are then allowed to reanneal. After washing and incubation in fluorescently labeled affinity reagents, a discrete fluorescent signal is visible at the site of probe hybridization. Application of FISH in biological studies has expanded rapidly sh:ce ihtroduction of the technique in the late 1970s :,2. Since then, over 200 reports have been made of the use of FISH in genomics, cytogenetics, prenatal and tumor diagnosis, radiation studies and basic biology. The expanding use of FISH is a result of several factors. (1) FISH has advantages over hybridization with isotopically labeled probes, including spatial resolution, speed and probe stability. (2) The sensitivity of FISH approaches that of isotopically labeled probes (2-5 kbp; see, for example, Refs 3-5), (3) A variety of probe-labeling schemes are available for simultaneous detection, in different colors, of two or more sequences in the same nucleus 6. (4) DNA sequences can be localized in targets ranging from metaphase cells spread on slides to interphase nuclei fixed in suspension to preserve their three-dimensional structure7. (5) Probe-labeling and fluorescent reagents are commercially available, making the FISH procedure straightforward and reliable. (6) The quality of fluorescence microscopes has steadily improved over the past decade. (7) The entire genome of a particular species, entire chromosomes, chromosomal subregions, or single-copy sequences can be specifically highlighted depending on the complexity of the probes used. (8) Hybridization of repetitive sequences can be suppressed by prehybridization of probes with unlabeled genomic DNA8. This is crucial for localization of unique sequences contained in large-insert probes, such as cosmids. An overview is presented here of the FISH technique, the variety of probe types available, and applications of FISH in cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping. Focus is limited to applications involving DNA sequence detection using a fluorescence microscope. Other important applications of nonisotopic in situ hybridization are arbitrarily excluded, such as ultrastructural studies through light and electron microscope analysis of probes carrying absorptive rather than fluorescent tags, studies of three-dimensional nuclear organization, viral or RNA sequence localization, and flow cytometric quantification of probe fluorescence.
Fluorescence in situ hybridization: applications in cytogenetics and gene mapping BARBARAJ. TIIASK Unique sequences, chromosomal subregions, or entire genomes can be specifically highlighted in metaphase or interphase cells by fluorescence in situ hybridization (FISH). This technique can be used to identify chromosomes, detect chromosomal abnormalities or determine the chromosomal location of specific sequences. FISHplays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping. Probe preparation Probes are labeled with reporter molecules and broken into 200~400 bp fragments, a size that maximizes specific hybridization and decreases background fluorescenceg. Although probes may be directly conjugated with fluorescent molecules 2, the most widespread approach is to label probes with reporter molecules that, after hybridization, bind fluorescent affinity reagents. Typical reporter molecules include biotin, digoxigenin, dinitrophenyl {DNP). aminoacetvlfluorene (AAF), mercury and sulfonate (reviewed in Ref. 10). The first three of these reporter molecules arc incorporated as labeled nucleotides using nick translation. Alternatively, probes can be labeled by PCR (polymerase chain reacti{}n~ amplification be:wecn known priming sequences or by RNA transcription from appropriate vectors in the f)rescncc of labclo_t nucleotides. AAE mercury and sulfonate are attached to DNA through chemical reactions. Size reduction is accomplished by nick translation itself or by sonicati()n after chemical modification. Hybridization Labeled probes are mixed in a hybridization l luffcJ containing formamide, salt and dextran sulfate. For repetitive probes, blocking DNA (e.g. sonicatcd herring testes DNA) is added to suppress nonspccifi, binding of probe to chromatin and glass. After denam ration, the mixture is applied directly t~} slides Incubation times as sh{)rt as 15 ,nin at 37°C arc st.ifficient to detect chronlosome-specific repetitive sequences. For unique sequence labeling using largeinsert probes, prehybridizati{m {)f the pr(}bc mixture i:: excess unlabeled genomic or Cotl DNA for up t() an hour may be necessary' to reduce repetitive sequence binding to the target s . Hybridization between target and probe then continues overnight at 37°C,
The technique Chromatin preparation and denaturation Cells are hypotonically swollen and fixed on slides by procedures developed for conventional banding analyses. Slides are incubated briefly at 70°C in a solution containing 70% formamide to melt the DNA into single strands, and are then fixed in cold ethanol to reduce strand reannealing before probe is added.
Fluorescent labeling After washing to remove mismatched or ~m hybridized probe molecules, slides are incubated in immunofluorescent reagents to produce a fluorescent signal at the sites of probe hybridization FI/l tv> order can be derived from the order of fluorescent dots if probes are >100 kbp apart and is easiest if detect muhiple chromosomal regions and to map sequences relative to each other in single cells. The probes are equidistant. most efficient means of image collection and handling An alternative system for high-resolution mapping have not yet been identified. Although digitization and uses pronuclei resulting after hamster eggs are fused in vitro with hamster or human sperm 40. After fixation computerized storage of images merits exploration, it may be unrealistic to expect that this approach will on slides, the chromatin in pronuclei disperses into a soon equal photography in terms of image size. re'; large (-50 gm in diameter) network of fibers, rather olution and speed of image handling. than a solid disk (10-20 btm in diameter) as in somatic The utility of FISH for cytogenetic analysis ~t nuclei. Figure 3C shows that hybridization sites of five metaphase and interphasc cells has n¢~t been cxplorccl probes mapping within 800 kbp on human chronicfully. The isolation, from critical chlomos¢,lnal lcgi¢~ns. some X are spread over a large distance in pronuclei of cosmid probes, which produce strong and discrete (tip to 20 gm). The distance between probes in prosignals, may alloy., more reliable and rapid detection
ECHNICAL ~'~ OCUS
F l u o r e s c e n t molecules can be deposited in chromatin at the sites of specific DNA sequences by use of fluorescence in situ hybridization (FISH). The technique is a simple one. DNA or RNA sequences are first labeled with reporter molecules. The probe and the target chromosomes or nuclei are denatured. Complementary sequences in the probe and target are then allowed to reanneal. After washing and incubation in fluorescently labeled affinity reagents, a discrete fluorescent signal is visible at the site of probe hybridization. Application of FISH in biological studies has expanded rapidly sh:ce ihtroduction of the technique in the late 1970s :,2. Since then, over 200 reports have been made of the use of FISH in genomics, cytogenetics, prenatal and tumor diagnosis, radiation studies and basic biology. The expanding use of FISH is a result of several factors. (1) FISH has advantages over hybridization with isotopically labeled probes, including spatial resolution, speed and probe stability. (2) The sensitivity of FISH approaches that of isotopically labeled probes (2-5 kbp; see, for example, Refs 3-5), (3) A variety of probe-labeling schemes are available for simultaneous detection, in different colors, of two or more sequences in the same nucleus 6. (4) DNA sequences can be localized in targets ranging from metaphase cells spread on slides to interphase nuclei fixed in suspension to preserve their three-dimensional structure7. (5) Probe-labeling and fluorescent reagents are commercially available, making the FISH procedure straightforward and reliable. (6) The quality of fluorescence microscopes has steadily improved over the past decade. (7) The entire genome of a particular species, entire chromosomes, chromosomal subregions, or single-copy sequences can be specifically highlighted depending on the complexity of the probes used. (8) Hybridization of repetitive sequences can be suppressed by prehybridization of probes with unlabeled genomic DNA8. This is crucial for localization of unique sequences contained in large-insert probes, such as cosmids. An overview is presented here of the FISH technique, the variety of probe types available, and applications of FISH in cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping. Focus is limited to applications involving DNA sequence detection using a fluorescence microscope. Other important applications of nonisotopic in situ hybridization are arbitrarily excluded, such as ultrastructural studies through light and electron microscope analysis of probes carrying absorptive rather than fluorescent tags, studies of three-dimensional nuclear organization, viral or RNA sequence localization, and flow cytometric quantification of probe fluorescence.
Fluorescence in situ hybridization: applications in cytogenetics and gene mapping BARBARAJ. TIIASK Unique sequences, chromosomal subregions, or entire genomes can be specifically highlighted in metaphase or interphase cells by fluorescence in situ hybridization (FISH). This technique can be used to identify chromosomes, detect chromosomal abnormalities or determine the chromosomal location of specific sequences. FISHplays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping. Probe preparation Probes are labeled with reporter molecules and broken into 200~400 bp fragments, a size that maximizes specific hybridization and decreases background fluorescenceg. Although probes may be directly conjugated with fluorescent molecules 2, the most widespread approach is to label probes with reporter molecules that, after hybridization, bind fluorescent affinity reagents. Typical reporter molecules include biotin, digoxigenin, dinitrophenyl {DNP). aminoacetvlfluorene (AAF), mercury and sulfonate (reviewed in Ref. 10). The first three of these reporter molecules arc incorporated as labeled nucleotides using nick translation. Alternatively, probes can be labeled by PCR (polymerase chain reacti{}n~ amplification be:wecn known priming sequences or by RNA transcription from appropriate vectors in the f)rescncc of labclo_t nucleotides. AAE mercury and sulfonate are attached to DNA through chemical reactions. Size reduction is accomplished by nick translation itself or by sonicati()n after chemical modification. Hybridization Labeled probes are mixed in a hybridization l luffcJ containing formamide, salt and dextran sulfate. For repetitive probes, blocking DNA (e.g. sonicatcd herring testes DNA) is added to suppress nonspccifi, binding of probe to chromatin and glass. After denam ration, the mixture is applied directly t~} slides Incubation times as sh{)rt as 15 ,nin at 37°C arc st.ifficient to detect chronlosome-specific repetitive sequences. For unique sequence labeling using largeinsert probes, prehybridizati{m {)f the pr(}bc mixture i:: excess unlabeled genomic or Cotl DNA for up t() an hour may be necessary' to reduce repetitive sequence binding to the target s . Hybridization between target and probe then continues overnight at 37°C,
The technique Chromatin preparation and denaturation Cells are hypotonically swollen and fixed on slides by procedures developed for conventional banding analyses. Slides are incubated briefly at 70°C in a solution containing 70% formamide to melt the DNA into single strands, and are then fixed in cold ethanol to reduce strand reannealing before probe is added.
Fluorescent labeling After washing to remove mismatched or ~m hybridized probe molecules, slides are incubated in immunofluorescent reagents to produce a fluorescent signal at the sites of probe hybridization FI/l tv> order can be derived from the order of fluorescent dots if probes are >100 kbp apart and is easiest if detect muhiple chromosomal regions and to map sequences relative to each other in single cells. The probes are equidistant. most efficient means of image collection and handling An alternative system for high-resolution mapping have not yet been identified. Although digitization and uses pronuclei resulting after hamster eggs are fused in vitro with hamster or human sperm 40. After fixation computerized storage of images merits exploration, it may be unrealistic to expect that this approach will on slides, the chromatin in pronuclei disperses into a soon equal photography in terms of image size. re'; large (-50 gm in diameter) network of fibers, rather olution and speed of image handling. than a solid disk (10-20 btm in diameter) as in somatic The utility of FISH for cytogenetic analysis ~t nuclei. Figure 3C shows that hybridization sites of five metaphase and interphasc cells has n¢~t been cxplorccl probes mapping within 800 kbp on human chronicfully. The isolation, from critical chlomos¢,lnal lcgi¢~ns. some X are spread over a large distance in pronuclei of cosmid probes, which produce strong and discrete (tip to 20 gm). The distance between probes in prosignals, may alloy., more reliable and rapid detection
