Making communications land - Are they received and understood as intended? we...
FISH FISH
1.
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.
4. I t i s a r el at i vel y new cyt ogenet i c
t echni que t hat al l ows a
cyt ogenet i ci st t o det er mi ne how many
copi es of a par t i cul ar chr omosome ar e
pr esent wi t hout havi ng t o go t hr ough
al l of t he st eps i nvol ved i n
pr oduci ng a kar yot ype.For example,
FISH analysis can quickly tell you how many number 21 chromosomes
are present, but it cannot tell you anything about the structure of those
chromosomes.
5. 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.
10. 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.
11. 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.
12. 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.
13. Chronic myeloid leukemia
• Cancer of White Blood Cells.
• Increased and unregulated groth of myeloid cells in
bone marow 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.
14. 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.
15. 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
16. Genetic diseases identified using FISH
Prader-Willi Syndrome
Prader-Willi syndrome is a complex
genetic condition that affects many
parts of the body. In infancy, this
condition is characterized by weak
muscle tone (hypotonia), feeding
difficulties, poor growth, and delayed
development. Beginning in childhood,
affected individuals develop an
insatiable appetite, which leads to
chronic overeating (hyperphagia) and
obesity. Some people with Prader-
Willi syndrome, particularly those with
obesity, also develop type 2 diabetes
mellitus (the most common form of
diabetes).
Prader-Willi syndrome is caused by the loss of function of genes in a particular region
of chromosome 15.
17. Angelman Syndrome
Angelman syndrome is a complex genetic disorder that primarily
affects the nervous system. Characteristic features of this condition
include delayed development, intellectual disability, severe speech
impairment, and problems with movement and balance.
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
18. Deletion detected by FISH
Deleted region of
chromosome 22-no flourescnt
signal
intact chromosome 22 giving a fluorescent
signal
19. • Miller-Dieker Syndrome
• Williams Syndrome de Williams
• Wolf-Hirschhorn Syndrome
• Smith-Magenis Syndrome
• Kallmann Syndrome etc. are the other methods.
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.
• This review aims to provide a detailed discussion of the CGH
technique, and to provide a protocol with an emphasis on crucial
steps.
22. HER-2/neu amplification detected by
fluorescence in situ hybridization in fine
needle aspirates from primary breast cancer.
The HER-2/neu gene is amplified in 20–30% of human
breast cancers and has been shown to have prognostic
and predictive value for treatment with
chemotherapy, hormone therapy and antibodies against
the HER-2/neudomain (trastuzumab).
