Molecular Cytogenetic Technique- (Fish)

• FISH is a cytogenetic technique that can be used to detect the presence or absence ofspecific DNA sequences (specific gene locus).
• fluorescent probes bind to only those parts of the chromosome with which they show a high degree of sequence similarity.like lncRNA, mRNA, or miRNA of interest
• FISH  can  be used for  rapid  identification  of chromosome during  interphase.
• FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine qtogenetics or identify extra material of unknown origin.
• it determines if a chromosome has a simple deletion or is involved in a subtle or complex rearrangement. In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers.
• This technique can be used on formalin-fixed paraffin embedded (FFPE) tissue, frozen tissues, fresh tissues, cells and circulating tumor cells.

Methodology:

• (FISH) analysis is performed by denaturing the double-stranded DNA in the fixed chromosomes on a microscope slide.
• Two fluorescently labeled DNA probes are used in combination to analyze each location.
• The first probe serves as a control and hybridizes with DNA on the target chromosome, The second probe hybridizes to a target location on the individual’s DNA sequence.
• When a deletion is present, the second probe will not hybridize and no fluorescence will be seen.
• A duplication will result in two fluorescent spots with the test probe.

Advantages of FISH

• FISH permits determination of the number and location of specific DNA sequences in human cells.
• FISH can be performed on metaphase chromosomes,as with G-banding, but can also be performed on cells not actively progressing through mitosis
• FISH performed on nondividing cells is referred to as interphase or nuclear FISH.

Disadvantages of FISH

• FISH requires a preselection of an informative molecular probe prior to analysis, So a prior knowledge of the anomaly is needed.

Uses  of  FISH:

• Detection of numeric abnormalities of chromosomes (aneuploidy). The demonstration of subtle microdeletions
• Detection of complex translocations not detectable by routine karyotyping
• For analysis of gene amplification e.g. HER2/NEU in breast cancer or N-MYC amplification in neuroblastomas
• For mapping newly isolated genesa of interest to their chromosomal loci

Chromosome  Painting:

• Is an extension of FISH, Probes are prepared for entire chromosomes.
• The number of chromosomes that can be detected simultaneouslyby chromosome painting is limited by the availability of fluorescent dyes.

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