Fluorescent In Situ Hybridization (FISH)

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COMPILED AND CIRCULATED BY DR. PRITHWI GHOSH, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE

BOTANY: SEM-VI, PAPER-DSE4T: ANALYTICAL TECHNIQUES IN PLANT SCIENCES, UNIT-1: FISH

**Fluorescent In Situ Hybridization (FISH)**

What is FISH?

Fluorescence in situ hybridization (FISH) is a kind of cytogenetic technique which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome. This technique provides a novel way for researchers to visualize and map the genetic material in an individual cell, including specific genes or portions of genes. It is an important tool for understanding a variety of chromosomal abnormalities and other genetic mutations. Different from most other techniques used for chromosomes study, FISH has no need to be performed on cells that are actively dividing, which makes it a very versatile procedure.

Fig. 1 Scheme of the principle of the FISH experiment to localize a gene in the nucleus.

How does FISH work?

FISH is useful, for example, to help a researcher identify where a particular gene falls within an individual's chromosomes. Here's how it works:

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• Make a probe complementary to the known sequence. When making the probe, label it with a fluorescent marker, e.g. fluorescein, by incorporating nucleotides that have the marker attached to them.

• Put the chromosomes on a microscope slide and denature them.

Denature the probe and add it to the microscope slide, allowing the probe to hybridize to its complementary site.

• Wash off the excess probe and observe the chromosomes under a fluorescent microscope.

The probe will show as one or more fluorescent signals in the microscope, depending on how many sites it can hybridize to.

Fig. 2 The five basic steps of FISH. (Oliveira and French 2005) What is FISH used for?

FISH is widely used for several diagnostic applications: identification of numerical and structural abnormalities, characterization of marker chromosomes, monitoring the effects of therapy, detection of minimal residual disease, tracking the origin of cells after bone marrow transplantation, identification of regions of deletion or amplification, detection of chromosome abnormalities in non-dividing or terminally differentiated cells, determination of lineage involvement of clonal cells, etc. Moreover, it has many applications in research: identification of non-random chromosome rearrangements, identification of translocation molecular breakpoint, identification of commonly deleted regions, gene mapping, characterization of somatic cells

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hybrids, identification of amplified genes, study the mechanism of rearrangements. FISH is also used to compare the genomes of two biological species to deduce evolutionary relationships.

Some more points

• Also used in germ cell or prenatal diagnosis of conditions such as aneuploidies.

• Use to detect and localize the presence and absence of the specific DNA sequences on chromosomes.

• FISH often used for finding specific features in DNA for use in genetic counselling, medicine, and species identification.

• FISH can also be used to detect and localize specific RNA target (mRNA, IncRNA, miRNA) in cells.

• Diseases that are diagnosed using FISH include Angelman syndrome, 22q13 deletion syndrome, acute lymphoblastic leukemia, Cri-du-chat, and Down syndrome.

• FISH can also be used to compare the genomes of two biological species, to deduce evolutionary relationships.

References

• https://www.creativebiomart.net/resource/principle-protocol-fluorescence-in-situ- hybridization-fish-protocol-342.htm

• https://www.genome.gov

• https://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-327/