o A powerful cytogenetic technique.
o It is used to detect localize the presence
or absence of specific DNA sequences on
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.
- Metaphase Chromosomes
- Interphase Nuclei
- Fixed Tissues
- Cells in culture
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.
Types of Probes
• Locus specific probes bind to a
particular region of a
• 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.
• Alphoid or centromeric repeat
probes are generated from
repetitive sequences found in
the middle of each
• 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
• Whole chromosome
probes are actually collections
of smaller probes, each of
which binds to a different
sequence along the length of a
• 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
example, when a piece of one
chromosome is attached to the
end of another chromosome.
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.
• In this translocation,
parts of chromosomes
9th and 22nd switch
• As a result , part of
BCR gene from
chromosome 22 is
fused with ABL gene
• BCR ABL fusion gene
prouct is a tyrosine
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
METAPHASE FISH INTERPHASE FISH
Acute lymphoblastic leukemia
• It is a cancer of white blood cells characterized by
• 12;21 translocation is most commonly found to
• This translocation results in TEL/AML1 gene fusion
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
Deletion detected by FISH
Deleted region of
chromosome 22-no flourescnt
intact chromosome 22 giving a fluorescent
22q13 Deletion Syndrome
• It is also known as Phelan-McDermid Syndrome.
• It is a genrtic disorder caused by deletions or rearrangements on
• 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
• Comparative genomic
hybridisation (CGH) is a technique
that permits the detection of
chromosomal copy number
changes without the need for cell
• 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
Comparative genomic hybridisation
• The green and red labelled DNA fragments compete for
hybridisation to their locus of origin on the
• 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.