Wednesday, 28 April 2021

FISH (Fluorescent In Situ Hybridisation) - Defination, Principle, Steps and Applications

FISH (fluorescent in situ hybridisation) is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.
  It is used to detect and localise the presence or absence of specific DNA sequences on chromosome.

Principle of FISH (fluorescent in situ hybridisation)

  FISH involves hybridising a fluorescent labelled DNA probe to denatured chromosomal DNA of metaphase chromosome, as well as I phase chromosomes.
   In FISH the DNA probe is either labelled directly by incorporation of fluorescent labelled nucleotides precursors or indirectly by incorporation of nucleotide precursors or containing a reporter molecule which after incorporation into DNA is then bound a pleasantly labelled affinity molecule.
   The position at which the probability to the chromosomal DNA is visualised by detecting the fluorescent signal emitted by the labelled DNA.

1). FISH analysis is performed by denaturing the double stranded DNA in fixed chromosome on a microscopic slide.

2). Once a denatured, two fluorescently labelled DNA probes are used to combination to analyse marker sequence location.

3). First probe serve as control and hybridize with DNA on target chromosome, but outside the target sequence.

4). The second probe hybridize  and locate the target sequence.
- If target sequence present, the probe will hybridize and fluorescence with colour other than control probe.
- If detection present, second probe will not hybridize and no fluorescent will seen in the fluorescent microscope.
- If the duplication two fluorescent spot will be seen with test probe.

Steps of FISH Assay

1). Preparation of Fluorescent Probes

- Fluorescent probes complementary sequences of target nucleic acid that are designed complementary to the sequence of inserts
- Size range from 20-40 base pair to 1000 bp.
- They are tagged with fluorescent dyes like biotin, fluracine and diaoxigenin.

2). Denaturation of Probe and the Target Sequence

- Denaturation is performed by heat or alkaline method on double stranded DNA in fixed chromosome on microscopic slide.

Hybridisation of Fluorescent
labelled probe with DNA

3). Hybridization of Probe and the Target Sequence

- Formation of duplex between two complementary nucleotide sequence
- It can be between DNA DNA, DNA RNA and RNA RNA.

4). Detection

Detection has Two types
a) direct labelling - lable is bound to the probe less sensitive
b) indirect labelling - require an additional step before detection.
Probe detected using antibodies conjugated to label like Alkaline Phosphate. It results into appreciation of signal.

5). Visualisation

- Fluorescent probe attaches to the target sequence during hybridization. This is visible light through a microscope with appropriate filters using fluorescent microscope.

Applications of FISH

•  FISH is used in germ cell or parental diagnosis of conditions such as aneuploidies.
• To detect and localised the presence and absence of the specific DNA sequence on chromosome and gene mapping.
• FISH also be used to detect and localise specific RNA target (mRNA and miRNA) in cells.
• Finding specific features in DNA for used in genetic counseling, medicine and species identification.
• Fish can be used to compare genomes of to biological species.
• Disease that are diagnosed using FISH include angelman syndrome, acute lymphoblastic leukaemia, cri-du-chat and down syndrome.
• The characterization of marker chromosome.
• Monitoring the success of bone marrow transplantation.


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