FISH (Fluorescent In-Situ Hybridization) is a technique that uses fluorescent probes that bind to complementary DNA sequences to localize specific genes or DNA sequences on chromosomes. It allows for direct visualization of the position of genetic markers on chromosomes. The FISH procedure involves denaturing chromosomes and probes, hybridizing the probes to target sequences, and examining slides under a fluorescence microscope. FISH has advantages like being rapid and allowing analysis of many cells, and has been improved over time, though accurate quantification remains challenging.

1. FLUORESCENT IN-SITU HYBRIDIZATION
TECHNIQUE
PRESENTED BY : DEV VRAT SHUKLA
DEPARTMENT OF BOTANY
UNIVERSITY OF ALLAHABAD

2. INTRODUCTION
A technique that hybridizes a DNA nucleic acid probe to a
target DNA sequence contained within a cell nucleus.
A variety of specimen types can by analyzed using FISH. The
intact cells are attached to a microscope slide using standard
cytogenetic methods.
FISH enables the position of a marker on a chromosome or
extended DNA molecule to be directly visualized
In FISH, the marker is a DNA sequence that is visualized by
hybridization with a fluorescent probe.

3. In situ hybridization intact chromosome is examined by probing
it with a labeled DNA molecule.
 The position on the chromosome at which hybridization occurs
provides information about the map location of the DNA
sequence used as the probe.
 DNA in the chromosome is made single stranded (‘denatured’).
 The standard method for denaturing chromosomal DNA
without destroying the morphology of the chromosome is to dry
the preparation onto a glass microscope slide and then treat with
formamide.

4. FISH

5. HISTORY OF DEVELOPMENT OF FISH
The abilities to detect specific molecular identities was first
demonstrated using antigen-antibody interactions.
 In 1977, the first antibody dependent fluorescent detection of nucleic
acid was achieved.
 The first application of fluorescent in situ detection come in 1980,
when RNA was directly labeled on the 3 end with fluorophore

6. PROBES
 Probe is a nucleic acid that
 can be labeled with a marker which allows
identification and quantitation
 will hybridize to another nucleic acid on the
basis of base complementarity

7. TYPES OF FISH PROBES
 Centromere
 Telomere
 Whole chromosome paint
 locus

8. TYPES OF PROBES
Centromeric (satellite)
probes
Locus specific probes
Whole chromosome painting probes

9. FISH PROCEDURE
Denature the chromosomes
Denature the probe
Hybridization
Fluorescence staining
Examine slides or store in the dark

10. Procedures Sample preparation and hybridization
Prepare slides with metaphase chromosomes or
interphase nuclei
Dehydrate in ethanol
Denature DNA at 70 oC
Denature labeled probe
Incubate at 37oC for 4-16 hours for
hybridization

12. HYBRIDIZATION
target DNA
probe
denaturation
hybridization

13. EpiFluorescent Microscope
CCD Camera
Filters
FISH Analysis
Software

14. ADVANTAGES OF FISH
Rapid
High efficiency of hybridization and detection
Lots of cells can be analyzed
Cells do not have to be replicating

15. PROBLEMS WITH IN SITU HYBRIDIZATION
 Permeabilization problems
Uneven cell penetration
 No signals
High amount of background autofluorescene

17. FISH using r-RNA targeted probes is the method of choice for all
studies in which exact cell numbers and cellular locations need to
be determined.
Development & design of more r-RNA targeted probes for novel
microorganisms in environment
The methodology is being continuously improved so far
however, microscopic analysis by FISH has not been automated
sufficiently which would be desirable in many investigations
Accurate quantification still remains a challenging task and
study needs careful controls.
Conclusion

18. REFERENCES
Elements of Biotechnology :- Gupta. P.K.
Crop improvement :- www.icar.org.in
www.dnalc.org
www.biocycleopidia.org
www.nottingham.ac

19. THANK YOU