This document provides an overview of fluorescence in situ hybridization (FISH). It discusses: 1. FISH is a cytogenetic technique that uses fluorescent probes to bind specifically to chromosomal regions with complementary DNA sequences, which can then be viewed under a fluorescent microscope. 2. FISH can be used to identify chromosomal abnormalities, assist with gene mapping, and determine ploidy. It has advantages over other techniques like being less labor intensive. 3. Common applications of FISH include detecting aneuploidies for prenatal diagnosis, locating specific DNA sequences on chromosomes, and identifying features in DNA for uses like genetic counseling.

1. Fluorescence in situ
Hybridization
PRESENTED BY : SHREYA LODH
UNDER THE GUIDANCE OF: DR.T.R.SHARMA
DR.R.K.CHAHOTA

2. INTRODUCTIONINTRODUCTION
 FISH is a cytogenetic technique that uses
fluorescent probes that bind to only those parts
of the chormosomes with a high degree of
sequences complementarity.
 Fluorescent probe-DNA complex can be studied
under fluroscent microscope.
 Developed by the biomedical researchers in
early’ 1980s.

3. Characteristics of FISH
FISH - a process which vividly paints
chromosomes or portions of chromosomes
with fluorescent molecules.
identifies the presence and location of a
region of DNA or RNA within morphologically
preserved chromosome preparations, fixed
cells or tissue sections

4. (FISH) contd.
Identifies chromosomal abnormalities
Aids in gene mapping, toxicological
studies, analysis of chromosome
structural aberrations, and ploidy
determination
Centromere regions stained brighter -
means they are rich in A-T bonds

5. Advantage of (FISH)
less labor-intensive method for
confirming the presence of a
DNA segment within an entire
genome than other
conventional methods like
Southern blotting

6. FISH: Limitations
Probe design requires knowledge of
specific chromosomal abnormalities to be
studied
Cutoff signals may be different among
laboratories
Processing errors, imperfect hybridization,
non-specific binding, photobleaching,
interobserver variability, and false positive
and negative results are possible

7. FISH Procedure
Denature the chromosomes
Denature the probe
Hybridization
Fluorescence staining
Detection or store in the dark

8. Probes
 Complementary sequences of target
nucleic acids
 Designed against the sequence of interest.
 Probes are tagged with fluorescent dyes
like biotin, fluorescein, Digoxigenin
 Size ranges from 20-40 bp to 1000bp.
 Stellaris(R) RNA FISH probes.

9. FISH Procedure
DETECTION
DENATURE OF
PROBE
DENATURE
OF DNA

10. Interpretation of FISH
Each fluorescently labeled probe that hybridizes
to a cell nucleus in the tissue of interest will
appear as a distinct fluorescent dot
 Diploid nuclei will have two dots
 If there is duplication in the region of
interest, the gain will result in more than
two dots
 If there is a loss in the region of interest,
one or zero dot will result

11. FISH Uses

12. APPLICATIONS
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 chormosomes.
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.

13. Differences between genomesDifferences between genomes
Major differences in the nature and amount of
repetitive DNA
• dpTa1 tandem repeat • 45S rDNA

14. FISH and Telomeres
Telomeric probes define the terminal
boundaries of chromosomes (5’ and 3’ ends)
Used in research of chromosomal
rearrangements and deletions related to cell
aging or other genetic abnormalities
Special telomeric probes specific to individual
chromosomes have been designed
Probe is based on the TTAGGG repeat
present on all human telomeres

15. FISH and Telomeres

16. FISH in Physical Mapping
A physical map is primarily based on the locations of
landmarks along a DNA molecule and units of distance are
expressed in base pairs.
Low Resolution Physical Mapping
 Cytogenetic map
 In situ hybridization

17. Procedure

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