Name: Fluorescent In-situ Hybridization: Principle, Use in Cytogenetics
Uploaded: 2021-04-07T12:12:00
Description: Learn by watching this video about Fluorescent In-situ Hybridization: Principle, Use in Cytogenetics at JoVE.com

Fluorescence In situ Hybridization or FISH is a technique used to detect the location of specific genes or parts of genes on chromosomes within fixed cells.
In this technique, a fluorescently labeled single-stranded DNA is used as a probe to bind parts of chromosomes that contain a complementary DNA sequence, referred to as the target.
First, cells are arrested in interphase or metaphase. The arrested cells are suspended in buffer solution and spread on a clean glass slide.
Next, the target DNA and the labeled probes are denatured with heat or chemicals. This step separates the double stranded DNA leaving both the probe and the target DNA as single-stranded sequences that are capable of base pairing.
Then, the probe and target DNA are mixed together and incubated overnight. This allows the probe to hybridize to its complementary sequence on the chromosomes.
Once hybridization is complete, the excess probe is washed off.
If the probe was labeled with a fluorophore, the locations of the hybridized DNA can be directly visualized with a fluorescent microscope.
If not, the hybridized DNA needs to be fluorescently labeled before visualization.&#160; To do so, the probe sequence must contain a modified nucleotide bonded to a hapten&#8211; a small molecule to which the fluorophore&#8212;such as a fluorescent antibody, can attach.
Such direct visualization of genes is very useful for cell-based diagnostic assays, where genetic abnormalities can be detected from any loss..., gain..., or rearrangement of chromosomes during cell division.

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	<li>Jiang, Jiming. &#34;Fluorescence in situ hybridization in plants: recent developments and future applications.&#34; Chromosome Research 27, no. 3 (2019): 153-165.</li>
	<li>Cui, Chenghua, Wei Shu, and Peining Li. &#34;Fluorescence in situ hybridization: cell-based genetic diagnostic and research applications.&#34; Frontiers in cell and developmental biology 4 (2016): 89.</li>
	<li>Frickmann, Hagen, Andreas Erich Zautner, Annette Moter, Judith Kikhney, Ralf Matthias Hagen, Henrik Stender, and Sven Poppert. &#34;Fluorescence in situ hybridization (FISH) in the microbiological diagnostic routine laboratory: a review.&#34; Critical reviews in microbiology 43, no. 3 (2017): 263-293.</li>
	<li>Prudent, Elsa, and Didier Raoult. &#34;Fluorescence in situ hybridization, a complementary molecular tool for the clinical diagnosis of infectious diseases by intracellular and fastidious bacteria.&#34; FEMS Microbiology Reviews 43, no. 1 (2019): 88-107.</li>
</ol>

fish - fluorescent in-situ hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques, CCD cameras, and sophisticated image-processing software, FISH signals can be interpreted with greater sensitivity and high specificity.
These technological advances have widened the scope of FISH applications from cytogenetics and gene mapping to the diagnosis of genetic and infectious diseases. High-resolution images obtained from FISH help identify chromosomal aberrations, such as common aneuploidy (abnormal number of chromosomes in a cell), microdeletion/microduplication syndromes (loss or gain of a chromosome), and subtelomeric rearrangements.
FISH is also used to detect the presence of microbial pathogens in human blood cells. In such diagnostic assays, short fluorescence-labeled DNA binds to the microbial 16s rRNA and can be visualized under a fluorescence microscope. It helps in the identification of the infectious agents at the genus or species level. FISH-based diagnostics allows for rapid identification of the pathogen, which is crucial for detecting slow-growing organisms like Mycobacteria - causative agent of tuberculosis or other microbes, such as Tropheryma whipplei that are difficult to culture.

Learn by watching this video about Fluorescent In-situ Hybridization: Principle, Use in Cytogenetics at JoVE.com