Download FISH

Biotechnology Students Training Course
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Advantages and Disadvantages of
conventional cytogenetic technique
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Advantages
Enable the entire genome to be viewed at one time.
Suitable when a specific anomaly is suspected ( e.g.
Philadelphia in CML ) and as a general diagnostic tool to
detect additional chr. Abnormalities commonly seen in
disease progression of CML.
Disadvantages
Detect major structural abnormalities
(one band = 6mb of DNA ~ 150 genes ).
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Labor intensive and highly dependent upon operator
experience and skills.
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Molecular cytogenetics:
Fluorescence in situ Hybridization (FISH)
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A process which distinctly paints and detects
RNA as well as DNA Structures, numbers and
location in place in the cell or in situ.
FISH may be used with:
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Morphologically preserved chromosome preparations
(Metaphase).
Fixed cells or tissue sections (Interphase)
Aids in gene mapping, toxicological studies,
analysis of chromosome structural aberrations,
and ploidy determination
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FISH Identifies chromosomal
abnormalities
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Indications for chromosomal
analysis
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Suspicion to concrete chromosomal abnormality
(concrete syndrome)
Multiple congenital anomalies or developmental
delay
Mental retardation
Gonadal dysgenesis
Infertility
Miscarriages
Delivery of dead fetus or death of a newborn child
Occurrence of certain malignancies
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What to do before indication of
chromosomal analysis?
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Exclude possibility of non-genetic affection
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Influence of teratogens or prenatal infections
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Complications during the birth (asphyxia, injuries of
the newborn child)
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Postnatal non genetic influence (injuries,
infections)
Exclude monogenic or multifactorial disorder
(= without abnormal chromosomal finding)
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In which conditions we have to
indicate FISH analysis?
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The material doesn't contain metaphase chromosomes
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Unsuccessful cultivation
It isn't possible to cultivate the tissue from patient (preimplantation
analysis, rapid prenatal examinations, examinations of solid
tumors or autopsy material)
Analysis of complicated chromosomal rearrangements
Identification of marker chromosomes
Diagnosis of submicroscopic (cryptic) chromosomal
rearrangements
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Microdeletion syndromes
Amplification of oncogenes and microdeletion of tumor-suppressor
genes in malignancies
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Tissue samples for FISH analysis
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Peripheral blood
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Fibroblasts from skin biopsy
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Epithelial cells from buccal smear
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Bone marrow (hemoblastosis)
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Solid tumor biobsies
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How to take a blood for FISH
analysis?
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Disinfect the skin with alcohol
(70% ethanol)
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Use tube with heparin or lithiumheparin to prevent blood clotting
(metaphase preparations).
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EDTA tube may also be used
(Interphase preparations).
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FISH Procedure
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Slides preparation from cultured (Metaphase) or
uncultured (Interphase) cells and fixed sing standard
cytogenetic prosedure.
Fixed cells are exposed to a probe (60-200–kb
fragment of DNA attached covalently to a fluorescent
molecule).
Denature the chromosomes
Denature the probe
Hybridization: The probe will hybridize or bind to its
complementary sequences in the cellular DNA
Fluorescence staining
The bound probe can be visualized under a
fluorescent microscope in the nucleus of the cell
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Locus specific probes
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Probes are designed to be specific to a particular
chromosome or chromosomal regions
A probe to any unique region on a chromosome should yield
an image of two signals per nucleus
A deletion or duplication of
the region that is hybridized
to the probe will result in a
nucleus with only one signal
or more than two signals,
respectively.
Multiple probes spanning
large regions are used to
detect regional deletions.
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Green: Chromosome 13
Red: Chromosome 21
Normal
Down syndrome
Buccal Cells from Swab
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Dual fusion probes
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Sometimes called dual-colored
probes
0.8–1.5 Mb in size
Designed to bind to regions
spanning the breakpoint of both
translocation partners.
A translocation will be observed
as a signal from both the
translocation junction and the
reciprocal of the translocation
junction; e.g., t(9;22) and t(22;9)
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Dual color break-apart probes
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0.6–1.5 Mb
Lowers background as well as identifies translocation
events
Designed to bind to the
intact chromosome flanking
the translocation
breakpoint.
When a translocation
occurs, the two probes
separate
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Centromeric probes (CEPs)
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Designed to hybridize to highly repetitive alpha
satellite sequences surrounding centromeres.
These probes detect aneuploidy of any
chromosome.
Combinations of centromeric probes and
region-specific probes are often used to
confirm deletions or amplifications in specific
chromosomes.
Addition of a CEP to dual color probes serves
as a control for amplification or loss of one of
the chromosomes involved in the translocation.
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This combination of CEP and dual color probes
comprises a tricolor probe
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Telomeric probes
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DNA probes specific to the telomeres of all human
chromosomes.
Useful for the detection of chromosome structural
abnormalities such as cryptic translocations or small
deletions that are not easily visualized by standard
karyotyping.
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Advantages of Interphase FISH
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Interphase cells for FISH do not require culturing
of the cells and stimulating division to get
metaphase spreads
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200–500 cells can be analyzed microscopically
using FISH
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interphase FISH is faster than methods using
metaphase cells
valuable for analysis of cells that do not divide well in
culture, including fixed cells.
the sensitivity of detection is higher than that of
metaphase procedures, which commonly examine 20
spreads.
Monitor recurrent or residual disease in BMT pt.
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Metaphase FISH
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Uses fluorescent probes that bind
to metaphase chromosomal
regions or to whole chromosomes.
Whole chromosome paints:
Probes that cover the entire
chromosome, are valuable for
detecting small rearrangements
that are not apparent by regular
chromosome banding.
Telomeric and centromeric probes
are also applied to metaphase
chromosomes to detect
aneuploidy and structural
abnormalities
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Spectral karyotyping (SKY) and multiple
fluoeescent hybridization (M-FISH)
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By mixing combinations of
five fluors and using special
imaging software, can
distinguish all 23
chromosomes by
chromosome specific colors.
This type of analysis can be
used to detect abnormalities
that affect multiple
chromosomes as is
sometimes found in cancer
cells or immortalized cell
lines.
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SKY
Advantages:
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Mapping of chromosomal breakpoints.
Detection of subtle translocations.
Identification of marker chromosomes, homogeneously
staining regions,and double minute chromosomes.
Characterization of complex rearrangements.
Disadvantages:
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Very expensive equipments.
The technique is labor intensive.
Dose not detect structural rearrangements within
single chromosome.
Low resolution (up to 15 mb ).
Specific, not a screening method.
a
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limitations of FISH
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The inability to identify chromosomal changes other than
those at the specific binding region of the probe.
Preparation of the sample is critical in interphase FISH
analysis
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to permeabilize the cells for optimal probe target interaction
to maintain cell morphology.
Cannot detect small mutations.
Miss Uniparental disomy.
Miss Inversions.
Probes are not yet commercially available for all
chromosomal regions
Relativelly expensive
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Diagnostic Potential For Karyotype and
FISH For Selected Disorders
Condition
Locus
studied
Karyotype
Disease
specific FISH
Telomere FISH
Aneuploidy
various
~100%
Not detected
Detected by
karyotype
Large deletions, large
dupllications, translocation of
large segments
various
~100%
Not detected
Detected by
karyotype
Cryptic
Rearrangements of telomeres
various
Not
detected
Not detected
~100%
1p36 deletion
1p36.3
Few
~99%
>95%
Wolf-Hirschhorn
4p16.3
Most
~99%
>95%
Cri-du-chat
5p15.2
Most
~99%
>95%
Williams-Beuren
7q11.2
Almost none
~99%
Not detected
Prader-Willi
15q11-q13
Unreliable
~70%
Not detected
Angelman
15q11-q13
Unreliable
~70%
Not detected
Miller-Dieker lissencephaly
17p13.3
Few
>90%
Smith-Magenis
17p11.2
Some
>95%
Not detected
Velocardiofacial/DiGeorage 1
22q11.2
Rarely
>95%
Not detected
Some detected
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Trisomy 21
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One of the most common chromosomal
abnormalities in live born children
Causes Down syndrome, a particular
combination of phenotypic features that includes
mental retardation and characteristic facies
(distinctive facial expressions associated with the
condition).
Molecular analysis has revealed that the
21q22.1-q22.3 region appears to contain the
gene(s) responsible for the congenital heart
disease observed in Down syndrome.
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Trisomy 13
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Also called Patau syndrome
Is a chromosomal condition that is associated
with severe mental retardation and certain
physical abnormalities.
The critical region has been reported to
include 13q14-13q32 with variable expression,
gene interactions, or interchromosomal
effects.
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Trisomy 18
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Causing Edwards syndrome is the second most
common autosomal trisomy after trisomy 21.
Characterized by severe psychomotor and
growth retardation, microcephaly,
microphthalmia, malformed ears, micrognathia or
retrognathia, microstomia, distinctively clenched
fingers, and other congenital malformations.
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X-Y related syndromes
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Turner syndrome:
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Metafemales or triple-X females:
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Inherit three X chromosomes; their genotype is XXX or more rarely
XXXX or XXXXX.
Klinefelter syndrome:
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Occurs when females inherit only one X chromosome; their
genotype is X0.
Males inherit one or more extra X chromosomes; their genotype is
XXY or more rarely XXXY, XXXXY, or XY/XXY mosaic.
XYY syndrome:
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Males inherit an extra Y chromosome; their genotype is XYY.
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Poseidon™ Repeat Free™ Chromosome
13/21, X/Y/18 specific DNA Probes
Vial 1
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Critical region 1 (red):
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Critical region 2 (green):
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The 21q specific DNA probe is direct-labeled with PlatinumBright550.
The 13q14 specific DNA probe is direct-labeled with
PlatinumBright495.
Vial 2
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Critical region 3 (blue):
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Critical region 4 (green):
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The 18 SE DNA probe is direct-labeled with PlatinumBright415.
The X SE DNA probe is direct-labeled with PlatinumBright495.
Critical region 5 (red):
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The Y SE DNA probe is direct-labeled with PlatinumBright550.
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Intended use
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The probes are optimized to detect copy
numbers of chromosome 21, 13, 18, X and Y
To be used with cultured or uncultured
amniotic or WBCs slide preparations.
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Chromosome 13/21 specific probe
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A dual-color assay to detect gains of
chromosome 21 and 13.
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Trisomy 21  (3R2G)
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Trisomy 13  (2R3G)
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3 red signal at the 21q22 region
2 green signals for chromosome 13
3 green signals at the 13q14 region
2 red signals for chromosome 21
Normal 13, 21  (2R2G)
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2 single color red (R) and green (G)
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Chromosome X/Y/18 specific probe
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A triple-colour assay to detect gains or losses of
chromosome X, Y and or 18.
Turner syndrome  one green signal only at Xcen.
Meta-Females (or Triple-X females)  3 or more green
signals at Xcen.
Klinefelter  2 or more green and 1 red signal.
XYY males  one green and two red signals.
normal X  2 single green signals
Normal male  one green and one red signal
Trisomy 18  3 blue signals at 18 cen.
normal chromosome 18  2 single blue signals