Chromosome Analysis

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Chromosome Analysis Karyotyping
process of preparing chromosomes for analysis
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Winners in triplicate
Carol Greider Elizabeth Blackburn Jack
Szostak
2009 Nobel Prize
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On Christmas Day in 1984, Greider then
Blackburn's graduate student saw the first
evidence that this enzyme, which Greider and
Blackburn named telomerase, was responsible for
constructing telomere DNA
--Telomerase provides a platform enabling DNA
polymerases to copy the entire length of the
chromosome without missing the ends. Greider and
Blackburn also showed that telomerase contains
a key RNA sequence that acts as a template
for the telomere DNA, which attracts proteins to
form a protective cap around the ends of the
DNA strands
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Int. J. Cancer 122, 14 (2008)
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Int. J. Cancer 122, 14 (2008)
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Int. J. Cancer 122, 14 (2008)
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Int. J. Cancer 122, 14 (2008)
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Chromosomal imbalances in oral squamous cell
carcinoma. Examination of 31 cell lines and
review of the literature
Oral Oncol. 2008 April 44(4) 369382.
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Karyotyping

1. Cell line identification
2. Cell line characterization
3. Relating the species and sex they were derived
4. Distinguishing between normal and malignant cells
5. Identification of aberrant chromosome
6. Embryonic stem cell characterization

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Karyotype G-banding Technique for
producing banding patterns in eukaryotic
chromosomes
Dark band , stained strongly with
giemsa stain or acredine
a. Heat hydrolysis
b. Trypsin treatment
c. Giemsa at pH 9.0 R- banding
Reverse banding ( Telomere bandinn)
stain weakly with giemsa or
acridine
pretreatment with BaOH or NaOH followed by heat
and salt
C-banding C (centromere or constitutive
heterochromatin)
stains the heterochromatin in the centromeres
chromosomes 1,
9, and 16 Q- banding Quinacrine
stain orange, fluorescent bands

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G-banding
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G banding
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Q- banding fluorescent bands
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Primary muscle cell culture
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C-banding stains the heterochromatin in the
centromeres
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Chromosome preparations Materials culture of
cells in log phase colcemid, 10-5 M in
PBSA PBSA Trypsin( 0.25) Hypotonic solution
0.04M KCl, 0.025M sodium citrate Acetic methanol
fixative( glacial acetic acid Methanol 1 3 )
Giemsa stain ( stock diluted in buffer water pH
6.8-7.2)
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Giemsa A polychromatic blood stain Stain nucleus
pink cytoplasm pale gray-blue
nucleoli dark blue
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• Chromosome preparation
• Culture in log phase
• Add clocemid 1x 10-5M in 1 100 vol ( final
  1x10-7M) or 100ug final concentration
• After 4-6 hrs, remove medium gently, and add
  0.25 trypsin, and incubate culture for 10 min
• Harvest cells by centrifugation, discard
  supernatant
• Resuspend cell in 5 ml hypotonic solution (KCl)
  and incubate 37oC, 15 min, varied for cell types
  ( do not exceed 15 min)
• Add equal volume of freshly prepared 1ml Acetic
  Methanol fixative solution, centrifuge 1200rpm,
  10 min
• Discard supernatant, vortex cell , and add 1ml
  Acetic Methanol fixative solution slowly
• Drop the solution from 30cm height on to the cold
  slide, tilt the slid and spread the solution
• Dry over the slides over a beaker as it boiling(
  or in hot oven 62oC, exam the slides under
  microscope , and prepare more slides
• Stain the cells with Giemsa

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• Stainning with Giemsa
• Immerse the slide in stain for 2 min
• 2. Place the dish into the water and allow the
  surplus stain to overflow from the top of the
  slide dish
• 3. Displace the remaining stain with running
  water

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