Fishy

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Fishy

  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. 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.
  4. 4. General schematic diagram of FISH
  5. 5. 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
  6. 6. Tagging of probes
  7. 7. 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.
  8. 8. • 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.
  9. 9. • 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.
  10. 10. 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.
  11. 11. 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.
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. Deletion detected by FISH Deleted region of chromosome 22-no flourescnt signal intact chromosome 22 giving a fluorescent signal
  16. 16. 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
  17. 17. • 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
  18. 18. • 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.
  19. 19. Schematic diagram of CGH and SKY
  20. 20. Presented by : Name : ANJALI BAJAJ Roll No :1754

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