This presentation provides an insight into the fundamentals of in situ hybridization (ISH), especially fluorescence in situ hybridization. It is ideal for classroom lecture.

1. FISHFluorescence in situ Hybridization
Presented By
Amartya Pradhan
Division of Biotechnology
Netaji Subhas University of Technology, New Delhi

2. First of all, what on earth is
ISH(in situ hybridization) ?(on site)

3. History of ISH
1953 - Watson and Crick
demonstrated how the two
strands of DNA are hold by
hydrogen bonding (secretly,
thanks to Chargaff for giving us
the concept of base pairing).
1969 - Gall & Pardue performed
the first in situ hybridization
experiment.
1991 - First application of FISH to
plant cytogenetics by Leitch et al.
1
2
3
Blame Them!
Mary-Lou Pardue Joseph G. Gall
(Founder of Modern Cell Biology)

4. Principle of ISH
• In situ hybridization is a technique used to find the
precise location of DNA sequences on chromosomes.
• It is based on the complementary region between the
bases of the target DNA segment and its reporter
molecule.
• ISH is a biological assay (like ELISA, PCR) for molecular
diagnosis. It is used to detect chromosomal abnormalities
(like down’s syndrome) during prenatal tests.

5. Take a note!
• A labeled RNA or DNA probe can be used to hybridize to
a known target mRNA or DNA sequence within a sample.
• This labeled RNA or DNA reporter/probe (antigen-, radio-,
or fluorescent-labeled) can then be detected by using an
antibody, autoradiography or fluorescence microscopy to
detect the label on the probe.
• The probes can therefore be used to detect expression of
a gene of interest and the location of the mRNA

6. Take it easy…

7. NOT THIS FISH
Let’s discuss
FISHFaster.
More specific.
Better sensitivity.
Versatile.
Forgive my Bengali instinct

8. Cytogeneticists can now go for "FISH-
ing"
• Today, most in situ hybridization procedures
use fluorescent probes to detect DNA
sequences, and the process is commonly
referred to as FISH (fluorescence in
situ hybridization).
• Principle same as that of ISH.
• Use of a fluorescent labeled probe
differentiates FISH from ISH; it is a part of
ISH.

10. The principles of FISH.
(A) The basic elements are a DNA probe
and a target sequence.
(B) Before hybridization, the DNA probe
is labelled indirectly with a hapten (left
panel) or directly labelled via the
incorporation of a fluorophore (right
panel).
(C) The labelled probe and the target
DNA are denatured to yield single-
stranded DNA. (D) They are then
combined, which allows the annealing of
complementary DNA sequences.
(E) If the probe has been labelled
indirectly, an extra step is required for
visualization of the non-fluorescent
hapten that uses an enzymatic or
immunological detection system. Finally,
the signals are evaluated by

11. Applications
• To detect chromosomal aberrations (additions, deletions, insertions,
inversions, copy number, translocations).
• Used in prenatal diagnosis (of Down syndrome etc.) and cancer diagnosis (to
detect TATAs and TSTAs).

12. Applications (continued)
• Used for gene mapping.
• Used to monitor disease progression (we can check expressions of proteins
in situ).
• Karyotyping (easier interpretation and better resolution because of
chromosome painting; we can colour any segment of chromosome with any
colour!). Types of chromosome painting:
 Whole chromosome painting probe (WPP)
 Chromosome arm painting probes (APP)
 Chromosome terminal band painting probes (TPP)
 Chromosome enumeration probes (CEP; paints near centromere)

13. Match the Following (based on the type of chromosome painting)
A B
C D
1. Chromosome enumeration probes
2. Whole chromosome painting probe
3. Chromosome arm painting probes
4. Chromosome terminal band painting probes

14. Match the Following (Answers)
A B
C D
1. Chromosome enumeration probes
2. Whole chromosome painting probe
3. Chromosome arm painting probes
4. Chromosome terminal band painting probes
(Answers)
A – 2
B – 4
C – 1
D – 3

15. References
1. Ryan Bishop; Applications of fluorescence in situ hybridization (FISH) in detecting
genetic aberrations of medical significance, Bioscience Horizons: The International
Journal of Student Research, Volume 3, Issue 1, 1 March 2010, Pages 85–
95, https://doi.org/10.1093/biohorizons/hzq009
2. https://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-
fish-327
3. https://www.slideshare.net/vbiogenex/fluorescent-insitu-hybridization-fish
4. https://en.wikipedia.org/wiki/In_situ_hybridization

16. Questions are appreciated