Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH) is a molecular technique designed to visualize and map specific genes on chromosomes within cells. This method serves as a pivotal diagnostic tool for identifying various genetic disorders and types of cancer. FISH utilizes fluorescently labeled probes that bind to complementary DNA sequences, allowing for the rapid detection of target DNA in a sample. The technique can be performed on different types of samples, including cells, tissues, and tumors, using various preparation methods to achieve high resolution. Probes can target specific genes, repetitive chromosome sequences, or entire chromosomes, making FISH versatile in genetic research and clinical applications.
FISH plays a significant role in prenatal and postnatal genetic testing, cancer cytogenetics, and the study of chromosomal structures and rearrangements. It is instrumental in diagnosing conditions such as Down syndrome and various cancers, including leukemia and breast cancer. Additionally, FISH has applications in microbial ecology for identifying microorganisms in various environments. Although modern advancements have led to the development of microarrays that often replace FISH, this technique remains crucial for understanding genetics and its implications in health and disease.
Fluorescence in situ hybridization (FISH)
SIGNIFICANCE: Fluorescence in situ hybridization (FISH) is a technique used to visualize and map the location of a specific gene on the chromosome in a cell. FISH is a powerful tool used to diagnose various genetic disorders and different forms of cancer.
Probes and Hybridization
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 hybridization. The 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 alphoid or centromeric repeat probe binds to the repetitive sequences found in the 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.
Target Chromosome Preparation
FISH can be performed on cells, tissues, and solid tumors. The different types of target chromosome preparations are preparation, 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.
Technique
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 FISH.
Applications
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. A wide array of FISH variants may be applied in such studies.
Impact
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 Charcot-Marie-Tooth syndrome, 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. In modern times, microarrays have replaced FISH.
Bibliography
Andreeff, Michael, and Daniel Pinkel, eds. Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications. New York: Wiley-Liss, 1999. Print.
Astbury, Caroline. Clinical Cytogenetics. Philadelphia: Saunders, 2011. eBook Collection (EBSCOhost). Web. 30 Nov. 2015.
Barbosa, Ana, Sonia Miranda, Nuno F. Azevedo, Laura Cerqueira, and Andreia S. Azevedo. "Imaging Biofilms Using Fluorescence In Situ Hybridization: Seeing Is Believing." Frontiers Cellular and Infection Microbiology, vol. 13, 2023, doi.org/10.3389/fcimb.2023.1195803. Accessed 4 Sept. 2024.
Dutra, Amalia. "Fluorescence In Situ Hybridization (FISH)." National Human Genome Research Institute, 3 Sept. 2024, www.genome.gov/genetics-glossary/Fluorescence-In-Situ-Hybridization. Accessed 3 Sept. 2024.
"Fluorescence In Situ Hybridization Fact Sheet." National Human Genome Research Institute, 16 Aug. 2020, www.genome.gov/about-genomics/fact-sheets/Fluorescence-In-Situ-Hybridization. Accessed 4 Sept. 2024.
Haimovich, Gal, ed. Fluorescence In Situ Hybridization (FISH): Methods and Protocols. Humana Press, 2024.
Liehr, Thomas, ed. Fluorescence In Situ Hybridization (FISH): Application Guide. New York: Springer, 2009. Print.
Speicher, Michael R., and Nigel P. Carter. “The New Cytogenetics: Blurring the Boundaries with Molecular Biology.” Nature Reviews: Genetics 6 (2005): 782–792. Print.
Zneimer, Susan Mahler. Cytogenetic Abnormalities: Chromosomal, FISH, And Microarray-Based Clinical Reporting. Chichester: Wiley, 2014. eBook Collection (EBSCOhost). Web. 30 Nov. 2015.