o A powerful cytogenetic technique.
o It is used to detect localize the presence
or absence of specific DNA sequences on
c...
FISH Targets
- Metaphase Chromosomes
- Interphase Nuclei
- Fixed Tissues
- Cells in culture
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
cop...
How does FISH work?
FISH is useful to help to identify where a particular gene
falls within an individual's chromosome.
A....
General schematic diagram of FISH
Directandindirectlabellingofprobes
DIRECT
FITC; rhodamine;Texas
red;cy2;cy3;cy5 and AMCA dyes
are most frequently used
IND...
Tagging of probes by nick translation
Types of Probes
Locus specific probes bind to a
particular region of a chromosome.
This type of probe is useful when
scien...
Alphoid or centromeric repeat
probes are generated from
repetitive sequences found in
the middle of each
chromosome. Resea...
Whole chromosome
probes are actually collections
of smaller probes, each of
which binds to a different
sequence along the ...
Chronic myeloid leukemia
• Cancer of White Blood Cells.
• Increased and unregulated groth of myeloid cells in
bone marow a...
Philadelphia chromosome
• In this
translocation, parts of
chromosomes 9th and
22nd switch places.
• As a result , part of
...
Detection of BCR ABL translocation. The green signal indicates the
presence of the BCR gene, red signals indicate the pres...
Genetic diseases identified using FISH
Prader-Willi Syndrome
Prader-Willi syndrome is a complex
genetic condition that aff...
Angelman Syndrome
Angelman syndrome is a complex genetic disorder that primarily
affects the nervous system. Characteristi...
Deletion detected by FISH
Deleted region of
chromosome 22-no flourescnt
signal
intact chromosome 22 giving a fluorescent
s...
• Miller-Dieker Syndrome
• Williams Syndrome de Williams
• Wolf-Hirschhorn Syndrome
• Smith-Magenis Syndrome
• Kallmann Sy...
• Comparative genomic
hybridisation (CGH) is a technique
that permits the detection of
chromosomal copy number
changes wit...
• The green and red labelled DNA fragments compete for
hybridisation to their locus of origin on the chromosomes.
• The gr...
HER-2/neu amplification detected by
fluorescence in situ hybridization in fine
needle aspirates from primary breast cancer...
Presented by :
Name : ANJALI BAJAJ
Roll No :1754
FISH FISH
FISH FISH
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FISH FISH

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FISH FISH

  1. 1. 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.
  2. 2. FISH Targets - Metaphase Chromosomes - Interphase Nuclei - Fixed Tissues - Cells in culture
  3. 3. 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.
  4. 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. 5. General schematic diagram of FISH
  6. 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. 7. Tagging of probes by nick translation
  8. 8. 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.
  9. 9. 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.
  10. 10. 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.
  11. 11. 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.
  12. 12. 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.
  13. 13. 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
  14. 14. 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.
  15. 15. 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
  16. 16. Deletion detected by FISH Deleted region of chromosome 22-no flourescnt signal intact chromosome 22 giving a fluorescent signal
  17. 17. • Miller-Dieker Syndrome • Williams Syndrome de Williams • Wolf-Hirschhorn Syndrome • Smith-Magenis Syndrome • Kallmann Syndrome etc. are the other methods.
  18. 18. • 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
  19. 19. • 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.
  20. 20. 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).
  21. 21. Presented by : Name : ANJALI BAJAJ Roll No :1754
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