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Fluorescence in situ Hybridization

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Fluorescence in situ Hybridization Fluorescence in situ Hybridization (FISH) FISH - a process which vividly paints chromosomes or portions of chromosomes with ... – PowerPoint PPT presentation

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Title: Fluorescence in situ Hybridization


1
Fluorescence in situ Hybridization
2
Fluorescence in situ Hybridization (FISH)
  • FISH - a process which vividly paints chromosomes
    or portions of chromosomes with fluorescent
    molecules
  • Opening picture - Human M-phase spread using DAPI
    stain

3
Fluorescence in situ Hybridization (FISH)
  • Identifies chromosomal abnormalities
  • Aids in gene mapping, toxicological studies,
    analysis of chromosome structural aberrations,
    and ploidy determination

4
Fluorescence in situ Hybridization (FISH)
  • Used to identify the presence and location of a
    region of DNA or RNA within morphologically
    preserved chromosome preparations, fixed cells or
    tissue sections

5
Fluorescence in situ Hybridization (FISH)
  • This means you can view a segment or entire
    chromosome with your own eyes
  • Was often used during M phase but is now used on
    I phase chromosomes as well

6
Fluorescence in situ Hybridization (FISH)
  • Advantage less labor-intensive method for
    confirming the presence of a DNA segment within
    an entire genome than other conventional methods
    like Southern blotting

7
FISH Procedure
  • Denature the chromosomes
  • Denature the probe
  • Hybridization
  • Fluorescence staining
  • Examine slides or store in the dark

8
FISH Procedure
9
FISH Uses
  • Detection of high concentrations of base pairs
  • Eg Mouse metaphase preparation stained with DAPI
    (a non-specific DNA binding dye with high
    affinity for A-T bonds)

10
FISH Uses
11
FISH Uses
  • Centromere regions stained brighter - means they
    are rich in A-T bonds
  • Also used in germ cell or prenatal diagnosis of
    conditions such as aneuploidies

12
FISH and Telomeres
  • Telomeric probes define the terminal boundaries
    of chromosomes (5 and 3 ends)
  • Used in research of chromosomal rearrangements
    and deletions related to cell aging or other
    genetic abnormalities

13
FISH and Telomeres
  • Special telomeric probes specific to individual
    chromosomes have been designed
  • Probe is based on the TTAGGG repeat present on
    all human telomeres

14
FISH and Telomeres
15
FISH and Telomeres
  • Application in cytogenetics - can detect
    submicroscopic deletions and cryptic
    translocations of genes associated with
    unexplained mental retardation and miscarriages

16
FISH and Telomeres - Medical from TelVysion DNA
website
  • A translocation between chromosomes 12 and 21
    with breakpoints at bands 12p13 (TEL)telomere
    and 21q22 (AML1) occurs in at least 25 percent of
    childhood B-cell acute lymphocytic leukemia

17
FISH and Telomeres - Medical
  • The LS1 TEL/AML1 ES Dual-Color Translocation
    Probe is designed to detect the TEL and AML1
    gene fusion
  • This fusion is undetectable by standard
    cytogenetics, but can be seen with FISH

18
FISH - Medical
  • FISH can be used in the study of transgenic
    animals (eg Polly)
  • Selective markers show if the human DNA was
    inserted successfully and pinpoint where the
    human DNA is
  • Transgenic Mouse

19
FISH - Medical
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