Fluorescence in Situ Hybridization (Fish)
Probes and Hybridization (Genetics & Inherited Conditions)
The two complementary deoxyribonucleic acid (DNA) strands are bound by hydrogen bonds. Heat and chemicals break the hydrogen bonds but they re-form when the conditions are favorable; this is the basis of nucleic acid hybridization. The probe is either tagged with biotin or digoxigenin, and they are detected by fluorophore conjugated streptavidin or antidigoxigenin antibody, respectively. Fluorophores are tagged directly to the probe, thus enabling rapid visualization of the target DNA. Fluorescent-labeled probes are safe, simple to use, and provide low background and high resolution. There are mainly three types of probes: The locus specific probe is used to locate the position of a particular gene on the chromosome, the centromeric repeat probe binds to the repetitive sequences found in the centromere of the chromosome, and the whole chromosome probe maps different regions along the length of any given chromosome. Thousands of bacterial artificial chromosome (BAC) clones obtained from the Human Genome Project are used as probes to map chromosomes. Probes are also available commercially.
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Target Chromosome Preparation (Genetics & Inherited Conditions)
FISH can be performed on cells, tissues, and solid tumors. The different types of target chromosome preparations are metaphase preparation, interphase preparation, and fibre FISH. In the metaphase preparation, the cells are captured in mitosis; the probes are large fragments that cover up to 5 megabases (Mb) and are used to map the entire chromosome. Interphase preparation is useful to study nondividing cells like those found in solid tumors. Hybridization occurs in the nucleus, thus enabling scientists to study the genome organization and location in its “natural” environment. The DNA is significantly less condensed in the interphase, which allows the probes to bind to their target DNA with greater resolution. The probes usually cover 50 kilobases (kb) to 2 megabases (Mb) of the chromosome. In fibre FISH, the interphase DNA is stripped of all proteins by either chemicals or mechanical shear. The released chromatin fibre can thus unfold and stretch into a straight line on a glass slide. This provides the highest resolution, from 5 kb to 500 kb. Fibre FISH is useful to study small rearrangements within the chromosome.
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Technique (Genetics & Inherited Conditions)
The target chromosome preparations are usually attached to a glass slide. The fluorescent-labeled probe and target chromosome DNA are denatured. The denatured probe is then applied to the target DNA and incubated for approximately twelve hours; this allows the probe to hybridize with its complementary sequence on the target chromosome DNA. The glass slide is washed several times to remove all unhybridized probes. The fluorescence in situ hybridization is then visualized by fluorescence microscopy. Advanced FISH techniques include multifluor (M) FISH, comparative genome hybridization (CGH), and microarray FISH.
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Applications (Genetics & Inherited Conditions)
In molecular biology, FISH is used to count the number of chromosomes in the cell. FISH visualizes chromosomal rearrangements such as translocation, inversion, and truncation. FISH is used to map genes and study the genome organization and structure in the cell. In the field of medicine FISH is used for prenatal and postnatal diagnosis of genetic disorders, cancer cytogenetics, and determination of infectious diseases. It plays a major role in understanding the chromosomal rearrangements that occurred during evolution and in developmental biology. FISH also plays a role in the field of microbial ecology. It is widely used for microorganism identification in drinking water and biofilms.
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Impact (Genetics & Inherited Conditions)
FISH played a major role in mapping genes on human chromosomes; this information was used during the annotation phase of the Human Genome Project. FISH is routinely used to diagnose and evaluate prognosis of cancers such as chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, bladder cancer, breast cancer, and ovarian cancer. It is useful in diagnosing genetic disorders such as Down syndrome. FISH is also used to diagnose diseases such as the Charcot-Marie-Tooth disease, Angelman syndrome, and Prader-Willi syndrome. It is used to screen donated blood for the presence of HIV-infected cells as well as in the clinical diagnosis of the infection.
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Further Reading (Genetics & Inherited Conditions)
Andreeff, Michael, and Daniel Pinkel, eds. Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications. New York: Wiley-Liss, 1999. This book covers the basic principles and techniques of FISH and describes in detail the applications of this technology to human cancer.
Liehr, Thomas, ed. Fluorescence In Situ Hybridization (FISH): Application Guide. New York: Springer, 2009. This book provides an overview about the principles and the basic techniques of FISH.
Speicher, Michael R., and Nigel P. Carter. “The New Cytogenetics: Blurring the Boundaries with Molecular Biology.” Nature Reviews: Genetics 6 (2005): 782-792. This review discusses the history of cytogenetics and the exciting advances in FISH.
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Web Sites of Interest (Genetics & Inherited Conditions)
National Institutes of Health. National Human Genome Research Institute. http://www.genome.gov/10000206
Scitable by Nature Education. Genetics: Fluorescence In Situ Hybridization (FISH). http://www.nature.com/scitable/topicpage/Fluorescence-In-Situ-Hybridization-FISH-327
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Fluorescence in Situ Hybridization (Fish) (World of Microbiology and Immunology)
Fluorescent in situ hybridization (FISH) is a technique in which single-stranded nucleic acids (usually DNA, but RNA may also be used) are permitted to interact so that complexes, or hybrids, are formed by molecules with sufficiently similar, complementary sequences. Through nucleic acid hybridization, the degree of sequence identity can be determined, and specific sequences can be detected and located on a given chromosome. It is a powerful technique for detecting RNA or DNA sequences in cells, tissues, and tumors. FISH provides a unique link among the studies of cell biology, cytogenetics, and molecular genetics.
The method is comprised of three basic steps: fixation of a specimen on a microscope slide, hybridization of labeled probe to homologous fragments of genomic DNA, and enzymatic detection of the tagged probe-target hybrids. While probe sequences were initially detected with isotopic reagents, nonisotopic hybridization has become increasingly popular, with fluorescent hybridization now a common choice. Protocols involving nonisotopic probes are considerably faster, with greater signal resolution, and provide options to visualize different targets simultaneously by combining various detection methods.
The detection of sequences on the target chromosomes is performed indirectly, commonly with biotinylated or digoxigenin-labeled probes detected via a fluorochrome-conjugated detection reagent, such as an antibody conjugated with fluorescein. As a result, the direct visualization of the relative position of the probes is possible. Increasingly, nucleic acid probes labeled directly with fluorochromes are used for the detection of large target sequences. This method takes less time and results in lower background; however, lower signal intensity is generated. Higher sensitivity can be obtained by building layers of detection reagents, resulting in amplification of the signal. Using such means, it is possible to detect single-copy sequences on chromosome with probes shorter than 0.8 kb.
Probes can vary in length from a few base pairs for synthetic oligonucleotides to larger than one Mbp. Probes of different types can be used to detect distinct DNA types. PCR-amplified repeated DNA sequences, oligonucleotides specific for repeat elements, or cloned repeat elements can be used to detect clusters of repetitive DNA in heterochromatin blocks or centromeric regions of individual chromosomes. These are useful in determining aberrations in the number of chromosomes present in a cell. In contrast, for detecting single locus targets, cDNAs or pieces of cloned genomic DNA, from 100 bp to 1 Mbp in size, can be used.
To detect specific chromosomes or chromosomal regions, chromosome-specific DNA libraries can be used as probes to delineate individual chromosomes from the full chromosomal complement. Specific probes have been commercially available for each of the human chromosomes since 1991.
Any given tissue or cell source, such as sections of frozen tumors, imprinted cells, cultured cells, or embedded sections, may be hybridized. The DNA probes are hybridized to chromosomes from dividing (metaphase) or non-dividing (interphase) cells.
The observation of the hybridized sequences is done using epifluorescence microscopy. White light from a source lamp is filtered so that only the relevant wavelengths for excitation of the fluorescent molecules reach the sample. The light emitted by fluorochromes is generally of larger wavelengths, which allows the distinction between excitation and emission light by means of a second optical filter. Therefore, it is possible to see bright-colored signals on a dark background. It is also possible to distinguish between several excitation and emission bands, thus between several fluorochromes, which allows the observation of many different probes on the same target.
FISH has a large number of applications in molecular biology and medical science, including gene mapping, diagnosis of chromosomal abnormalities, and studies of cellular structure and function. Chromosomes in three-dimensionally preserved nuclei can be "painted" using FISH. In clinical research, FISH can be used for prenatal diagnosis of inherited chromosomal aberrations, postnatal diagnosis of carriers of genetic disease, diagnosis of infectious disease, viral and bacterial disease, tumor cytogenetic diagnosis, and detection of aberrant gene expression. In laboratory research, FISH can be used for mapping chromosomal genes, to study the evolution of genomes (Zoo FISH), analyzing nuclear organization, visualization of chromosomal territories and chromatin in interphase cells, to analyze dynamic nuclear processes, somatic hybrid cells, replication, chromosome sorting, and to study tumor biology. It can also be used in developmental biology to study the temporal expression of genes during differentiation and development. Recently, high resolution FISH has become a popular method for ordering genes or DNA markers within chromosomal regions of interest.
See also Biochemical analysis techniques; Biotechnology; Laboratory techniques in immunology; Laboratory techniques in microbiology; Molecular biology and molecular genetics