Fluorescence In Situ Hybridization (FISH) | Learn Science at...

来自 : 发布时间：2024-04-30

By:Clare O\'Connor, Ph.D.(Biology Department, Boston College) 2008Nature Education Citation:O\'Connor,C.(2008)Fluorescence in situ hybridization (FISH).Nature Education1(1):171Cytogenetics entered the molecular era with the introductionof in situ hybridization, a procedurethat allows researchers to locate the positions of specific DNA sequences onchromosomes. Since the first in situ hybridizationexperiments in 1969 (Gall Pardue, 1969), many variations of the procedurehave been developed, and its sensitivity has increased enormously. Today, most in situ hybridization procedures usefluorescent probes to detect DNA sequences, and the process is commonlyreferred to as FISH (fluorescence in situhybridization). A variety of FISH procedures are available tocytogeneticists, who use them to diagnose many types of chromosomalabnormalities in patients. The success of FISH, and all other methods of in situ hybridization, depends on theremarkable stability of the DNA double helix. In 1953, James Watson and Francis Crick described theextensive network of hydrogen bonds that hold together the two antiparallelstrands in the DNAdouble helix (Watson Crick, 1953).Today, even schoolchildren know that adenine on one DNA strand binds to thymineon the complementary DNA strand, and that cytosine likewise binds to guanine. Becauseof the many hydrogen bonds formed between these bases, the double helix is aremarkably stable structure. Moreover, if the hydrogen bonds that hold thehelix together are broken with heat or chemicals, the helix is able to re-formwhen conditions become more favorable. This ability of the DNA helix to re-form,or renature, provides the basis for molecular hybridization. In molecular hybridization, a labeled DNA or RNA sequence isused as a probe to identify or quantify the naturally occurring counterpart ofthe sequence in a biological sample. In the 1960s, researchers Joseph Gall andMary Lou Pardue realized that molecular hybridization could be used to identifythe position of DNA sequences in situ(i.e., in their natural positions within a chromosome). In fact, in 1969, thetwo scientists published a landmark paper demonstrating that radioactive copiesof a ribosomal DNA sequence could be used to detect complementary DNA sequencesin the nucleus of a frog egg. Since those original observations, manyrefinements have increased the versatility and sensitivity of the procedure tothe extent that in situ hybridizationis now considered an essential tool in cytogenetics.Figure 1:Principles of fluorescence in situ hybridization (FISH).(a) The basic elements of FISH are a DNA probe and a target sequence. (b) Before hybridization, the DNA probe is labeled by various means, such as nick translation, random primed labeling, and PCR. Two labeling strategies are commonly used: indirect labeling (left panel) and direct labeling (right panel). For indirect labeling, probes are labeled with modified nucleotides that contain a hapten, whereas direct labeling uses nucleotides that have been directly modified to contain a fluorophore. (c) The labeled probe and the target DNA are denatured. (d) Combining the denatured probe and target allows the annealing of complementary DNA sequences. (e) If the probe has been labeled indirectly, an extra step is required for visualization of the nonfluorescent hapten that uses an enzymatic or immunological detection system. Whereas FISH is faster with directly labeled probes, indirect labeling offers the advantage of signal amplification by using several layers of antibodies, and it might therefore produce a signal that is brighter compared with background levels. 2005 Nature Publishing Group Speicher, M. R. et al. The new cytogenetics: blurring the boundaries with molecular biology. Nature Reviews Genetics 6, 784 (2005). All rights reserved.Soon after Gall and Pardue\'s work, fluorescent labels quickly replaced radioactive labels in hybridization probes because of their greater safety, stability, and ease of detection (Rudkin Stollar, 1977). In fact, most current in situ hybridization is done using FISH procedures (Trask, 2002; Speicher Carter, 2005). Detecting a DNA sequence can be compared to looking for a needle in a haystack, with the needle being the DNA sequence of interest and the haystack being a set of chromosomes. This search is made much easier if the investigator has a powerful \"magnet\" in this case, a fluorescent copy of the DNA sequence of interest. Hybridization occurs when the \"magnet\" meets the \"needle\"; this requires both a probe and a target, as shown in Figure 1. In the figure, the probe sequence, often a piece of cloned DNA, is shown in red. The target DNA chromosomes on a glass slide is shown in blue (in the right column). Hydrogen bonds that join the two strands of the DNA helix are represented by black lines. The first step in the process is to make either a fluorescent copy of the probe sequence (Figure 1b, middle column) or a modified copy of the probe sequence that can be rendered fluorescent later in the procedure (Figure 1b, left column). Next, before any hybridization can occur, both the target and the probe sequences must be denatured with heat or chemicals (Figure 1c). This denaturation step is necessary in order for new hydrogen bonds to form between the target and the probe during the subsequent hybridization step. The probe and target sequences are then mixed together (Figure 1d), and the probe specifically hybridizes to its complementary sequence on the chromosome. If the probe is already fluorescent (middle column), it will be possible to detect the site of hybridization directly. In other cases (left column), an additional step may be needed to visualize the hybridized probe. Hybrids formed between the probes and their chromosomal targets can be detected using a fluorescent microscope. When investigators design a FISH experiment, they need to consider whether the sensitivity and resolution needed for the experiment lie within the technical limits of fluorescence microscopy. Sensitivity depends on the light-gathering ability of the particular microscope, which determines whether small target sequences, which are more difficult to see than large target sequences, can be detected. Resolution refers to the ability to distinguish between two points along the length of a chromosome. Ultimately, light microscopy cannot resolve objects that are separated by less than 200 250 nm, the lower limit of the visible light spectrum. With these technical limits in mind, investigators also need to consider the conformation of DNA within the chromosome. Metaphase chromosomes are thousands of times more compacted than interphase chromosomes, which in turn are at least ten times more compacted than naked DNA. (Remember that one 3.4 nm turn of the DNA helix corresponds to 10 base pairs of DNA.) When all these factors are considered together, investigators typically expect to obtain resolution in the range of megabases for positions on metaphase chromosomes and resolution in the range of tens of thousands of kilobases for interphase chromosomes.Figure 2:Cytogenetic analyses of sequence-integrated clones.(a) Using FISH, fluorescent signals are observed at cytogenetic bands (grey) where fragments of a sequence-tagged bacterial artificial chromosome hybridize (red). (b) A clone selected on the basis of band location is used in FISH analysis to map the breakpoint of a translocation involving chromosomes 11 and 19 in a patient with multiple congenital malformations and mental retardation. The clone spans the breakpoint on chromosome 19; thus, the red signal is split between the derivative 11 and derivative 19 chromosomes and is present on the normal chromosome 19.2001 Nature Publishing Group Cheung, V. G. et al. Integration of cytogenetic landmarks into the draft sequence of the human genome. Nature 409, 954 (2001). All rights reserved.FISH provides a powerful tool for identifying the location of a cloned DNA sequence on metaphase chromosomes. Figure 2a shows the results of a typical FISH experiment, in which a cloned DNA sequence was hybridized to normal metaphase chromosomes. Red bands are detected at hybridization sites on two homologous chromosomes, which can be identified by their characteristic banding patterns. Closer examination shows that each red band actually consists of two spots, corresponding to the two sister chromatids in a mitotic chromosome. A skilled cytogeneticist would be able to use these hybridization data together with the banding pattern to place the probe sequence within a few megabases of other known genes on the chromosome. Historically, FISH and other in situ hybridization results played a primary role in mapping genes on human chromosomes. Results from these experiments were collected and compiled in databases, and this information proved useful during the annotation phase of the Human Genome Project (HGP). Now that the HGP is complete, investigators rarely use in situ hybridization simply to identify the chromosomal location of a human gene. (In species for which the genome has not been sequenced, however, FISH and related in situ hybridization methods continue to provide important data for mapping the positions of genes on chromosomes.) Currently, human FISH applications are principally directed toward clinical diagnoses.FISH and other in situhybridization procedures are important in the clinical diagnosis of variouschromosomal abnormalities, including deletions, duplications, andtranslocations. Figure 2b shows one example in which investigators used FISHtogether with standard karyotyping toanalyze a patient translocation. The hybridization probe corresponded to asegment of chromosome 19 that was suspected to include the translocationbreakpoint. Three areas of hybridization are apparent in the fluorescent image.One spot corresponds to the patient\'s normal copy of chromosome 19 (nl19), andthe other two spots correspond to the altered, or derived (der), versions ofchromosomes 11 and 19 that were produced during the translocation. Thus,investigators were able to use the data both to narrow down the breakpointregion on chromosome 19 and to identify the second chromosome involved in thetranslocation.The hybridization probe used in Figure 2b was one ofthousands of bacterial artificial chromosome (BAC) clones fromthe HGP that have been made available to the scientific community. Today,cytogeneticists are able to use extensive HGP clone resources to preciselyidentify the sites of chromosomal rearrangements that appear in karyotypes. Infact, a consortium of scientists has mapped over 7,000 DNA clones from the HGPto specific bands on human chromosomes (BAC Research Consortium, 2001). Atleast one clone is available for every megabase segment of chromosomal DNA.(The only exception is the Y chromosome, because it is relatively gene-poor.) Figure 3:Spectral karyotyping and multicolor-FISH paint each human chromosome in one of 24 colors.Cytogenetic localization of DNA sequences with fluorescence in situ hybridization (FISH). 2014 Panels a) and b) modified from 2000 Cambridge University Press. McNeil, N. Ried, T. Novel molecular cytogenetic techniques for identifying complex chromosomal rearrangements: technology and applications in molecular medicine. Expert Reviews in Molecular Medicine (online: September 2000). All rights reserved. The detection of chromosome rearrangements with site-specific probes (Figure 2b) can be a lengthy endeavor, especially if complex rearrangements have occurred or if the rearranged regions are difficult to identify by their banding patterns in a karyotype. Fortunately, cytogeneticists now have the option of using multifluor FISH, or spectral karyotyping, to quickly scan a set of metaphase chromosomes for potential rearrangements (Speicher et al., 1996; Schrock et al., 1996). Multifluor FISH generates a karyotype in which each chromosome appears to be painted with a different color. Each \"paint\" is actually a collection of hybridization probes for sequences that span the length of a particular chromosome. With multifluor FISH, investigators first prepare a collection of DNA sequences to be used as probes for each chromosome. In Figure 3a, the probe chromosomes have been physically separated from one another by flow cytometry. (Today, investigators would probably use commercially available DNA collections for each chromosome.) In the next step, the DNA samples are labeled with combinations of fluorochromes that produce a unique color for each chromosome. (The Cot-1 DNA step in the figure removes repetitive DNA sequences [e.g., centromeric DNA] that would bind to all chromosomes.) The fluorescent hybridization probes are then combined with and hybridized to metaphase chromosomes. Figure 3b shows images of interphase and metaphase chromosomes as they would appear through a microscope after hybridization. To human eyes, several of the metaphase chromosomes appear to have the same color, but digital processing of the image would distinguish spectral differences between the chromosomes. A normal human chromosome (Figure 3b) will have a uniform color along its length, but a rearranged chromosome will have a striped appearance. Although chromosome paints allow rapid assessment of large chromosomal changes in metaphase spreads, the resolution of the method is limited. Thus, while chromosome painting allows investigators to quickly identify chromosomes involved in translocations and to identify large deletions and/or duplications, small deletions and duplications will not be detectable. If investigators need more detailed information about the actual sequences involved in chromosomal rearrangements, they need to follow up with site-specific probes, as previously described (Figure 2).Since the introduction of FISH, cytogeneticists have beenable to analyze interphase chromosomes as well as the metaphase chromosomesused in karyotypes (Trask, 2002). Thisoffers a real practical advantage, in that cells do not need to be cultured forseveral days or weeks before chromosomes can be prepared for analysis. Inaddition, FISH can be used to analyze chromosomes from specimens such as solidtumors, which are of great clinical interest but do not divide frequently. Anotheruseful feature of FISH is that researchers are able to simultaneously monitormultiple sites if the hybridization probes have been labeled with differentfluorophores.Figure 4 shows two examples of how interphase FISH can be used to diagnose chromosomeabnormalities. Figure 4a shows an interphase nucleus from a patient with Charcot-Marie-Tooth disease(CMT) type 1A (Lupski et al., 1991). CMTtype 1A is a relatively common neurological condition caused by a duplicationin a gene on chromosome 17 that encodes one of the proteins in the myelinsheath that surrounds nerve axons. In Figure 4a, the patient\'s cell has beenhybridized with a red-labeled probe corresponding to a sequence within theduplicated region, along with a green probe corresponding to a sequence onchromosome 17 that lies outside of the duplicated region. From the two greensignals, it is possible to locate two copies of chromosome 17 within thenucleus. One chromosome has the normal configuration, while the second, der(17),contains the duplicated region, which is evident from two nearby red signals. Thefigure also serves to illustrate another important feature of interphase FISH.Because interphase chromatin is about 10,000 times less compacted than mitoticchromatin, it is possible to resolve the duplicated regions on der(17) asdiscrete points. This small duplication would have been difficult to resolve inmitotic chromosomes.Figure 4b shows a FISH analysis that was used to detect thepresence of a chromosomal translocation in a patient suffering from chronicmyelogenous leukemia (Tkachuk et al.,1990). In most cases of this disease, a segment of chromosome 9 that containsthe ABL proto-oncogene fuses with thebreakpoint cluster region (BCR) onchromosome 22 during a reciprocal translocation. The derived chromosome 22, order(22), also known as the Philadelphiachromosome, contains a BCR-ABL fusiongene in which the powerful BCRpromoter drives synthesis of the ABL oncogenetranscript, leading to cancer. Figure 4b demonstrates that BCR-ABL fusions can be readily identified by FISH when a green-labeledhybridization probe flanking BCR isapplied together with a red-labeled probe flanking ABL. In this image, the normal copies of chromosomes 9 and 22 aredetected as red and green spots, respectively. On the other hand, the Philadelphia chromosomeis visible as a complex fused spot, which appears to have a central yellowregion with red and green subregions on either side. (In fluorescencemicroscopy, yellow is indicative of very close proximity of red and greenprobes, such that they appear to overlap.) The intricate substructure of thefused spot is detectable in interphase chromosomes, but it would not beresolved in a similar FISH analysis of metaphase chromosomes. Thus, two-colorinterphase FISH provides a sensitive method for analyzing chromosome fusionevents without the need for a prior cell culture.Another research application of interphase FISH makes use ofchromosome-specific paints to obtain information about the organization ofchromosomes within the nucleus. Figure 2a (upper left) shows an interphasenucleus that has been stained with chromosome-specific paints. One can see fromthe figure that the chromosomes occupy distinct territories withinthe nucleus. By creatively combining chromosome-specific probes withgene-specific probes and antibodies, investigators can use FISH to provideexciting new insights about nuclear architecture.Exciting new applications of FISH that extend its rangecontinue to be developed. For example, cytogeneticists now use comparativegenomic hybridization todetect quantitative differences, like copy number variations, in thechromosomes of their patients. Recently, investigators have also been able toincrease the resolution of FISH by using stretched chromatin fibers (Parra Windle, 1993) or microarrays as the target. With tools such as these,cytogenetics has been able to move from studying whole chromosomes on themacroscopic scale, to studying the DNA of which these chromosomes consist.BAC Resource Consortium. Integration of cytogenetic landmarks into the draft sequence of the human genome. Nature 409, 953 958 (2001) (link to article)Gall, J. G., Pardue, M. L. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proceedings of the National Academy of Sciences 63, 378 383 (1969) Lupski, J. R., et al. DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 66, 219 232 (1991) doi:10.1016/0092-8674(91)90613-4Parra, I., Windle, B. High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature Genetics 5, 17 21 (1993) doi:10.1038/ng0993-17 (link to article)Rudkin, G. T., Stollar, B. D. High resolution of DNA-RNA hybrids in situ by indirect immunofluorescence. Nature 265, 472 474 (1977) doi:10.1038/265472a0 (link to article)Schrock, E., et al. Multicolor spectral karyotyping of human chromosomes. Science 273, 494 497 (1996) doi:10.1126/science.273.5274.494Speicher, M. R., Ballard, S. G., Ward, D. C. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nature Genetics 12, 368 375 (1996) doi:10.1038/ng0496-368 (link to article)Speicher, M. R., Carter, N. P. The new cytogenetics: Blurring the boundaries with molecular biology. Nature Reviews Genetics 6, 782 792 (2005) doi:10.1038/nrg1692 (link to article)Tkachuk, D. C., et al. Detection of BCR-ABL fusion in chronic myelogenous leukemia by in situ hybridization. Science 250, 559 562 (1990) doi:10.1126/science.2237408Trask, B. J. Human cytogenetics: 46 chromosomes, 46 years and counting. Nature Reviews Genetics 3, 769 778 (2002) doi:10.1038/nrg905 (link to article)Watson, J. D., Crick, F. H. C. Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid. Nature 171, 737 738 (1953) doi:10.1038/171737a0 (link to article) Topic rooms within Chromosomes and Cytogenetics Close You have authorized LearnCasting of your reading list in Scitable. Do you want to LearnCast this session? Yes No