fluorescence in situ hybridization

1. BSBT 521
Molecular and Immunodiagnostics
Session 2019-20
FISH- Fluorescence in
situ hybridization
Made by – Nitya Bansal
ASU2016010200104
IBT- 8th semester
Submitted to – Dr. Zeeshan Nazm

2. Introduction
FISH- Fluorescence in-situ hybridization is a molecular
cytogenetic technique that tags genetic material with
fluorescent molecules. It is a technique for mapping the
location of genes onto chromosomes.
https://www.genome.gov/sites/default/files/tg/en/illustration/fluorescence_in_situ
_hybridization_fish.jpg

3. History
The technique first developed in 1982. But its use and application came
more into play after 1990 because by then easily accessed methods of
probe generation and detection and many other techniques were
introduced.

4. Procedure
I. Denature the
chromosomes
II. Denature the
probe
III. Hybridization
IV. Fluorescence
staining
V. Detection
https://upload.wikimedia.org/wikipedia/commons/e/e6/FISH_%28technique%29.gif

5. Principles of FISH
a) The basic elements are a DNA probe
and a target sequence.
b) Before hybridization, the DNA probe is
labeled indirectly with a hapten or
directly labeled via the incorporation
of a fluorophore.
c) The labeled 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.
f) Finally, the signals are evaluated by
fluorescent microscope
https://image2.slideserve.com/4147595/principles-of-fish-l.jpg

6. Probes
Probe is a nucleic acid that:
can be labeled with a marker which allows
identification and quantification
will hybridize to another nucleic acid on the basis of
base complementarity
A part of DNA (or RNA) that is complementary to
certain sequence on target DNA (i.e. DNA of the patient)
Plasmid, phage DNA, cosmid (or combination of phage
and plasmid DNA
PCR-product (amplification of certain segment of
chromosomal DNA).

7. Types of probes
Scientists use three different types of FISH probes, each of
which has a different application:
1) Locus specific probes bind to a particular region of a
chromosome. This type of probe is useful when scientists
have isolated a small portion of a gene and want to
determine on which chromosome the gene is located, or
how many copies of a gene exist within a particular
genome.
2) Alphoid or centromeric repeat probes are generated
from repetitive sequences found in the middle of each
chromosome These probes can also be used in
combination with "locus specific probes" to determine
whether an individual is missing genetic material from a
particular chromosome.
3) Whole chromosome probes are actually collections of
smaller probes, each of which binds to a different
sequence along the length of a given chromosome.

8. https://image.slidesharecdn.com/presentationpptfish-170815075553/95/fish-fluorescence-in-
situ-hybridization-20-638.jpg?cb=1551454493

9. Hybridizer
Fluorescent microscope
https://encrypted-tbn0.gstatic.com/images?q=tbn%3AANd9GcQALRN_S2HOFeoJDS97cNqO46hpKRqwYc-
vk8qpQ1xwzgbVvbtZ

10. Interpretation of results
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
 If a small deletion is present in the region
complementary to the probe, the probe will not
hybridise
 If a duplication is present, more of the probe is able to
hybridise.

11. https://www.researchgate.net/publication/333203522/figure/fig3/AS:761165923753988@1558487506856/a-
Fluorescent-in-situ-hybridization-FISH-technique-applied-in-a-normal-pattern-of.png

12. Applications
Diagnostic:
• The identification of specific
chromosome abnormalities.
• The characterization of marker
chromosomes.
• Interphase FISH for specific
abnormalities in cases of failed
cytogenetic.
• Monitoring disease
progression
• Monitoring the success of
bone marrow transplantation .
Research:
• The identification of new non-
random abnormalities (by M-
FISH or SKY).
• Gene mapping
• Identification of regions of
amplification or deletion by
CGH.
• The identification of
translocation breakpoints
• The study of 3D chromosome
organization in interphase
nuclei.

13. Advantages
Rapid technique and large number of cells can
be scored in a short period.
Efficiency of Hybridization and deletion is
high. Sensitivity and specificity is high.
Cytogenetic data can be obtained from non-
dividing or terminally differentiated cells.
Cytogenetic data can be obtained from poor
samples that contain too few cells for routine
cytogenetic analysis.

14. Limitations
1. Restricted to those abnormalities that can be
detected with currently available probe.
2. Only one or a few abnormalities can be assessed
simultaneously.
3. Due to Failure to detect signal FISH is higher sensitive
for trisomy but less sensitive foe detecting
chromosome loss or deletion.
4. Cytogenetic data can be obtained only for the target
chromosomes thus FISH is not a good screening tool
for cytogenetically heterogeneous disease.
5. Requires fluorescence Microscopy and an image
analysis system.

15. References
• https://highered.mheducation.com/sites/9834092339/
student_view0/chapter18/fish.html
• https://www.hindawi.com/journals/bmri/2015/461524
/
• http://citeseerx.ist.psu.edu/viewdoc/download?doi=10
.1.1.112.5331&rep=rep1&type=pdf
• https://www.ncbi.nlm.nih.gov/pubmed/20809300
• https://academic.oup.com/biohorizons/article/3/1/85/
228880/Applications-of-fluorescence-in-situ-
hybridization

16. Thank you