2. o A powerful cytogenetic technique.
o It is used to detect localize the presence
or absence of specific DNA sequences on
chromosomes.
o Exploits the ability of single stranded
DNA to anneal to complementary DNA.
o Uses fluorescent probes.
o Fluorescence microscopy detects the
presence of fluorescent probes.
o It is a powerful technique used in the
detection of chromosomal abnormalities.
Fluorescence in situ hybridization (FISH) is a molecular
diagnostic technique utilizing labeled DNA probes to
detect or confirm gene or chromosome abnormalities.

3. FISH Targets
- Metaphase Chromosomes
- Interphase Nuclei
- Fixed Tissues
- Cells in culture

4. How does FISH work?
FISH is useful to help to identify where a particular gene
falls within an individual's chromosome.
A. The first step is to prepare short sequences of single-stranded
DNA that match a portion of the gene. These are called probes.
B. The next step is to label these probes by attaching one of a
number of colors of fluorescent dye.
C. DNA is composed of two strands of complementary molecules
that bind to each other like chemical magnets.
D. When a probe binds to a chromosome, its fluorescent tag
provides a way to see its location using fluorescent microscope.

5. General schematic diagram of FISH

6. Directandindirectlabellingofprobes
DIRECT
FITC; rhodamine;Texas
red;cy2;cy3;cy5 and AMCA dyes
are most frequently used
INDIRECT
biotin;digoxigenin & DNP reprtr
molecules are frequently used

7. Tagging of probes

9. Types of Probes
• 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.

10. • Alphoid or centromeric repeat
probes are generated from
repetitive sequences found in
the middle of each
chromosome.
• Researchers use these probes
to determine whether an
individual has the correct
number of chromosomes.
• 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.

11. • Whole chromosome
probes are actually collections
of smaller probes, each of
which binds to a different
sequence along the length of a
given chromosome.
• Using multiple probes labeled
with a mixture of different
fluorescent dyes, scientists are
able to label each chromosome
in its own unique color.
• The resulting full-color map of
the chromosome is known as a
spectral karyotype. Whole
chromosome probes are
particularly useful for
examining chromosomal
abnormalities, for
example, when a piece of one
chromosome is attached to the
end of another chromosome.

12. Chronic myeloid leukemia
• Cancer of White Blood Cells.
• Increased and unregulated growth of myeloid cells in
bone marrow and accumulation of these cells in blood.
• It is a type of first malignancy to be linked to a clear
genetic abnormality which is the chromosomal
translocation known as philadelphia chromosome.
• More common in males.

13. Philadelphia chromosome
• In this translocation,
parts of chromosomes
9th and 22nd switch
places.
• As a result , part of
BCR gene from
chromosome 22 is
fused with ABL gene
on chromosome.
• BCR ABL fusion gene
prouct is a tyrosine
kinase-remains
continuously on.

14. Detection of BCR ABL translocation. The green signal indicates the
presence of the BCR gene, red signals indicate the presence of
the ABL gene and the red-green fusion (yellow) signal confirms a
BCR/ABL translocation. The extra red signal confirms this is not a false
positive result.
METAPHASE FISH INTERPHASE FISH

15. Acute lymphoblastic leukemia
• It is a cancer of white blood cells characterized by
excessive lymphoblasts.
• 12;21 translocation is most commonly found to
be associated.
• This translocation results in TEL/AML1 gene fusion

16. DiGeorge and velo-cardio-facial Syndromes
It is caused by deletion of small piece of
long arm of chromosome 22 near the
middle at a location designated as
22q11.2

17. Deletion detected by FISH
Deleted region of
chromosome 22-no flourescnt
signal
intact chromosome 22 giving a fluorescent
signal

18. 22q13 Deletion Syndrome
• It is also known as Phelan-McDermid Syndrome.
• It is a genrtic disorder caused by deletions or rearrangements on
chromosome 22.
• The deletion occurs at the termainal end of the chromosome at the
locatoin designated q13.3.
• In this syndrome; deletion of SHANK3 gene is associated wid autism
spectrum disorder and schizophrenia

20. • Comparative genomic
hybridisation (CGH) is a technique
that permits the detection of
chromosomal copy number
changes without the need for cell
culturing.
• It provides a global overview of
chromosomal gains and losses
throughout the whole genome of a
tumour. Tumour DNA is labelled
with a green fluorochrome, which
is subsequently mixed (1:1) with
red labelled normal DNA and
hybridised to normal human
metaphase preparations.
Comparative genomic hybridisation

21. • The green and red labelled DNA fragments compete for
hybridisation to their locus of origin on the
chromosomes.
• The green to red fluorescence ratio measured along
the chromosomal axis represents loss or gain of
genetic material in the tumour at that specific locus.
• In addition to a fluorescence microscope, the
technique requires a computer with dedicated image
analysis software to perform the analysis.

22. Schematic diagram of CGH and SKY

23. Presented by :
Name : ANJALI BAJAJ
Roll No :1754