FISH is a molecular cytogenetic technique that uses fluorescent probes to bind specifically to parts of chromosomes. It is used to detect and localize DNA sequences on chromosomes. The FISH assay involves fixing cells, designing probes, denaturing DNA, hybridizing probes to complementary DNA, and using fluorescence microscopy to view results. FISH has applications like detecting Down syndrome and provides advantages over other methods like being less labor-intensive, though imperfect hybridization or non-specific binding can produce errors.

1. MUHAMMAD IKRAM
2018-MLT-007
Insta: 404_troubleshooter

2. Fluorescence in Situ
Hybridization
(FISH)

3. • 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 localize the presence or absence of specific DNA sequences on
chromosomes.

4. FISH Assay

5. The steps for FISH-assay are as follows:
1. Fixation of cell.
2. Probe designing.
3. Denaturation.
4. Hybridization.
5. Fluorescence microscopy.

6. Fixation
To removes cytoplas- mic material and makes the cell's nuclear DNA accessible to
the hybridization probes.

7. Design of Probes
• DNase (inducing random cuts/Nicks).
• Endonuclease enzymes.
(Double-stranded DNA) (Nicks)

8. Design of Probes
Labelling the probe:
• Nucleotide with florescent protein.
• Ligase seals ; nicks.
(Nucleotide bases with florescent protein) (Nicks are filled)

9. Denaturation
(Both the strands of DNA and
probe have denatured due
to high temperature)

10. Hybridization
(Attachment of probe to
its complementary
DNA strand)

11. Fluorescence Microscopy

12. Results

14. Applications

15. • Chorionic villus sampling (CVS) i.e. Down syndrome

16. Advantages
&
Limitations

17. • Advantages:
Less labor-intensive method than other conventional methods like southern blotting.
• Limitations:
Processing errors, imperfect hybridization, non-specific binding, photobleaching,
interobserver variability, and false positive and negative results are possible.