PART 2 Flashcards
(309 cards)
1
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VISUALIZING CHROMOSOMES
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2
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- CONVENTIONAL CYTOLOGICAL STAINS:
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FEULGEN, WRIGHT & HEMATOXYLIN
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• Feulgen
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Wright
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• FEULGEN STAIN (commonly used)
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→ can visualize DNA on tissue sections and in
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cells. This staining is the most used staining
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to highlight DNA in histology.
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• HEMATOXYLIN
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→ and eosin staining helps identify different
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types of cells and tissues and provides
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important information about the pattern
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shape
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and structure of cells in a tissue
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sample.
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- FLUORESCENT DYES
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• Quinacrine & Quinacrine mustard
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• Pattern: Q bands - 1st demonstrated by
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Caspersson
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Zech
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• QUINACRINE
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→ fluorescent staining method q banding
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is
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used to identify individual chromosomes
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and their structural anomalies
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given the
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resulting banding pattern. The
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characteristic banding pattern can be
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used to identify each chromosome
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accurately.
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3. CHEMICAL DYE
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• Giemsa stain (pre-treatment: trypsin)
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• Pattern: G bands
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4. HARSHER TREATMENT OF CHROMOSOMES
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Incubate in hot phosphate buffer (87ºC for 10 min
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then cooling to 70ºC) before Giemsa staining)
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• Pattern: R bands (can be visualized after staining
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w/ acridine orange)
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5. ALKALI TREATMENT
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• Centromere staining
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• Pattern: C bands
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TECHNIQUE PROCEDURE BANDING PATTERN
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G-BANDING o Proteolysis
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with Trypsin
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o Staining
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with
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Giemsa dye
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o Dark bands (A-T
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Rich)
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o Light Bands (G-C
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Rich)
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R-BANDING o Heat
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denature
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o Staining
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with
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Giemsa Dye
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o Dark bands (G-C
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Rich)
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o Light bands (A-T
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Rich)
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Q-BANDING o Staining
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with
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Quinacrine
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Mustard
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Dye
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o Dark
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bands/bright
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fluorescence (A-T
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Rich)
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o Light bands/dull
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(G-C Rich)
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C-BANDING o Denature
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with Barium
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Hydroxide
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o Staining
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with
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Giemsa Dye
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o Dark bands
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o (Constitute
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Heterochromatin)
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6. NUCLEOLAR ORGANIZING (NOR) STAINING
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• Silver nitrate: stain specifically at the constricted
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regions
or stalks
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chromosomes
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• are chromosomal regions crucial for the
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formation of the nucleolus.
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7. 4’6-DIAMIDINO-2-PHENYLINDOLE (DAPI)
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• Binds to the surface grooves of dsDNA
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• Fluoresces blue under UV light (353-nm
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wavelength)
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• Visualization of chromosomes & whole nuclei
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CHROMOSOME BANDING facilitates the:
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▪ Detection of deletions
insertions
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& other abnormalities
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▪ ID of distinct chromosomal locations
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DETECTION OF GENOME & CHROMOSOMAL
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MUTATIONS
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1. FLOW CYTOMETRY
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• Indirect method of detecting genome mutations
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or aneuploidy
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• Measuring DNA content of individual cells
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• Aneuploidy is reflected by a change in the
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amount of DNA
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2. KARYOTYPING
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• Direct method of detecting genome mutations
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or aneuploidy
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o observation of metaphase chromosome
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structure by arranging them according to
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size
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• Karyotype: chart that shows the complete set of
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chromosomes in a cell
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• Performed in light microscope
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Karyotypes:
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Can also detect chromosomal mutations:
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A. TRANSLOCATION
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• Exchange of genetic material between
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chromosomes
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TYPES DESCRIPTION
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a. Reciprocal Parts of 2
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chromosomes
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Exchange
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b. Robertsonian Movement of long
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arm of an
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acrocentric
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chromosome to the
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centromere of
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another
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acrocentric
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chromosome
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B. DELETION
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• Loss of chromosomal material
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C. INSERTION
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• Gain of chromosomal material
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D. INVERSIONS
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• Result from excision
flipping
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chromosomal material w/in the same
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chromosome
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Normal male karyotype
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Designated: 46
XY
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Aneuploidy involving Y
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chromosome disomy (XYY
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syndrome)
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Designated: 47
XYY
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Pericentric – include centromere in the inverted region
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Paracentric – involve sequences within 1 arm of the chromosome
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E. ISOCHROMOSOME
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Metacentric chromosome resulting from transverse splitting of centromere during cell division
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F. RING CHROMOSOME
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Results from deletion of genetic regions from ends of the chromosome and a joining of the ends to form a ring
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G. DERIVATIVE CHROMOSOME
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Translocated / otherwise rearranged parts from 2 or more unidentified chromosomes joined to a normal chromosome DID YOU KNOW…
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That the 1st chromosome mutations associated with human disease were visualized in the 1960s in leukemia cells?
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❖
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Peter Nowell and David Hungerford observed an abnormally small chromosome 22 in leukemia cells (Philadelphia chromosome)
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❖
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1972: Janet Rowley 1st described the translocation between chromosomes 8 & 21 in patients with acute myeloblastic leukemia
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❖
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Same year
she demonstrated that the Philadelphia chromosome was the result of a reciprocal exchange between chromosome 9 & 22 TERMS USED IN EXPRESSING
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Results of karyotyping analysis:
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Number of chromosomes per nucleus (normal = 46)
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Type of sex chromosomes (normal = XX or XY)
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Genetic abnormalities observed
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Normal karyotype:
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46
XX = female 46
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Example 1: 46
XX
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Meaning: A female with deletion in the long arm of
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chromosome 7 at region 1
band 3
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Example 2: 47
XX
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Meaning: A female with trisomy 21 (Down syndrome)
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Example 3: 46
XY
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Meaning: A male with a translocation between the short arms of chromosome 5 & 17 at region 1
band 3
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3. FLUORESCENCE IN SITU HYBRIDIZATION (FISH)
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Method widely used to detect proteins & nucleic acids
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Targets specific sequences of chromosomes w/ fluorescent probes
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o
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designed to hybridize to critical areas that are amplified/deleted/translocated/
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otherwise rearranged in disease states
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Requires fluorescence microscope & special filters
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Types: interphase FISH & metaphase FISH INTERPHASE FISH
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Used to study the genetic content & organization of non-dividing cells
particularly cells in the interphase stage of the cell cycle
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Bound probe is visualized under a fluorescent microscope as a point of fluorescent light in the nucleus of the cell
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Deletion = 1 signal
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Duplication = more than 2 signals
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Commonly used to study prenatal samples
tumors
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FISH analysis using centromeric probes for a normal diploid cell (left)
triploidy or trisomy (center)
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PROBES:
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a. Translocations/other rearrangements = probes of different color/signals
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Dual-color probes/dual-fusion probes: bind to regions spanning the breakpoint of translocations.
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Break-apart probes: bind to the intact chromosome flanking the translocation breakpoint.
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b. Centromeric (CEN) probe
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Hybridize to highly repetitive alpha satellite sequences surrounding centromeres
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Detect aneusomy of any chromosome
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+ region-specific probes = to confirm deletions / amplifications in specific chromosomes
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+ dual-color probes = tricolor probe
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o
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serves as a control for amplification/loss of 1 of the chromosomes involved in the translocation
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Example: IGH/MYC/CEP 8 Tri-color probes
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c. Telomeric probes
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Designed to specifically target & visualize the telomeric regions of chromosomes.
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Useful for detection of chromosome structural abnormalities (cryptic translocations/sub-telomeric deletions) that are not easily visualized by standard karyotyping.
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PROCEDURE:
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1. Preparation of sample
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Permeabilize the cells for optimal-target interaction & to maintain cell morphology
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Fresh interphase cells are incubated overnight (aging) after deposition on slides
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Cells are treated with protease and fixed with 1% formaldehyde
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Cells are dehydrated in graded concentration of ethanol
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Denaturation
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2. Quality of the probe
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Under a fluorescent microscope w/ appropriate color-distinction filters: signal should be bright
specific to the target in the cell nuclei
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3. Both probe & target must be denatured prior to hybridization
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1-10 μg of probe may be used in a hybridization volume of 3-10Μl
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Hybridization: 37ºC-42ºC in a humidified chamber
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Slides are cover-slipped & sealed
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4. Washing
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Rinsing off the unbound probe
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5. Microscopic analysis
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Sample is observed under fluorescent microscope
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Probe signals should be visible from entire intact nuclei
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Adequate number of cells must be visible METAPHASE FISH
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Allows analysis of small regions not visible by regular chromosome banding
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Probes that cover the entire chromosome/whole chromosome paints
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Combination of 5 fluors & using a special imaging software
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o
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spectral karyotyping can distinguish all 23 chromosomes by chromosome-specific colors (detect abnormalities that affect multiple chromosomes)
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Telomeric & centromeric probes are also used
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o
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detection of aneuploidy & other genomic mutations
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PROCEDURE:
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Culture of cells for 72 hours
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45 mins before harvesting
colcemid is added
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Cells are suspended in a hypotonic medium (0.075 M KCl) & fixed with methanol/acetic acid (3:1)
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Fixed cell suspension is applied to an inclined slide & allowed to dry
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2nd treatment w/ 70% acetic acid
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Under a phase contrast microscope
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o
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chromosome should appear well separated w/ sharp borders & cytoplasm should not be visible
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Dry the slide & proceed to hybridization
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MULTICOLOR FISH (QMFISH or M-FISH)
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• Simultaneous use of combinations of different
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locus-specific probes & chromosome paints
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• Uses multiple fluorescently labeled probes that
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target different chromosomal regions/genes
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• Identifies specific chromosomal regions based on
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the presence/absence of the probe color
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visualized with specific filters
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• May show cryptic translocations & insertions
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4. COMPARATIVE GENOME HYBRIDIZATION (CGH)
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• Detection of intrachromosomal
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amplifications/deletions
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• Test DNA is isolated & labeled along with a
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reference DNA
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• Fluorescent labels:
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→ Cy3 – fluoresces @ 550 nm “green”
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(reference DNA)
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→ Cy5 – fluoresces @ 650-667 nm “red” (test
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DNA)
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→ Cy3.5 – fluoresces in the red-orange
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region
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WHAT ARE THE CAUSES OF ABNORMALITIES?
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• Error in cell division
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o can result in cells w/ too few or too many
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copies of a chromosome
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• Maternal age
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o chromosomal errors can appear in eggs
