Human metaphase chromosomes
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Human Metaphase Chromosomes. Experiment Objectives. Preparing, staining and observing human metaphase chromosomes. Chromosome Morphology. Chromosomes are not visible under the light microscope in non-dividing cells (interphase cells).

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Human Metaphase Chromosomes

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Human metaphase chromosomes

Human Metaphase Chromosomes


Experiment objectives

Experiment Objectives

  • Preparing, staining and observing human metaphase chromosomes.

Mazen Zaharna Molecular Biology 1/2009


Chromosome morphology

Chromosome Morphology

  • Chromosomes are not visible under the light microscope in non-dividing cells (interphase cells).

  • As the cell begins to divide, the threads of chromatin (DNA-protein complex) in the nucleus begin to condense into multiple levels of coiled structures recognizable as chromosomes.

  • There are two modes of cell division:

    • mitosis and meiosis. Mitosis is responsible for the proliferation of body (somatic) cells,

    • whereas meiosis is responsible for the production of gametes.

  • Because mitotic cells are easy to obtain, morphological studies are generally based on mitotic metaphase chromosomes.

Mazen Zaharna Molecular Biology 1/2009


Cell division

Cell division

  • Cell division can be divided into:

    • Interphase,

    • Mitosis

      • Prophase,

      • Metaphase,

      • Anaphase,

      • Telophase.

    • Cytokinesis

Mazen Zaharna Molecular Biology 1/2009


Metaphase

Metaphase

  • At metaphase the chromosomes are at their most condensed state,

  • Spindle fibers attaching to the area of the centromere called the kinetochore, forming pole-chromosome fibers.

Mazen Zaharna Molecular Biology 1/2009


Chromosome analysis

Chromosome Analysis

  • The best mitotic stage for chromosome analysis is prometaphase or metaphase.

  • A typical metaphase chromosome consists of two arms separated by a primary constriction or centromere.

  • Each of the two sister-chromatids contains a highly coiled double helix of DNA.

Mazen Zaharna Molecular Biology 1/2009


Chromosome analysis1

Chromosome Analysis

  • Often the sister chromatids are so close to each other that the whole chromosome appears as a single rod-like structure

  • A chromosome may be characterized by its total length and the position of its centromere

Mazen Zaharna Molecular Biology 1/2009


Human metaphase chromosomes

Mazen Zaharna Molecular Biology 1/2009


Types of tissue

Types of Tissue

  • A variety of tissue types can be used to obtain chromosome preparations.

  • Some examples include peripheral blood, bone marrow, amniotic fluid and products of conception.

  • In the case of blood cell culture only cells that are actively dividing can be used for cytogenetic studies.

  • Normally only white blood cells are used for cytogenetic analysis.

  • Specific techniques differ according to the type of tissue used.

Mazen Zaharna Molecular Biology 1/2009


Overview of procedure

Overview of Procedure

  • Collection of blood

  • Cell culture

  • Stopping the cell division at Metaphase

  • Hypotonic treatment of red & white blood cells

  • Fixation

  • Slide preparation

  • Staining

Mazen Zaharna Molecular Biology 1/2009


1 collection of blood

1- Collection of blood

  • Draw 5 ml of venous blood into a sterile heparinized tube containing 0.1 ml of sodium heparin (500 units/ml).

Mazen Zaharna Molecular Biology 1/2009


2 cell culture

2- Cell Culture

  • Sterile technique must be used throughout the cell culture preparation, because it is possible to cause major contamination during this procedure

  • 70% of the problems are due to a lack of good sterile technique

  • Antibiotics do not eliminate problems of gross contamination which result from poor sterile technique or antibiotic-resistant mutants

  • Autoclaving renders pipettes, glassware, and solutions sterile

Mazen Zaharna Molecular Biology 1/2009


2 cell culture1

2- Cell Culture

Medium

  • Pipette 10 ml RPMI 1640 medium with L-Glutamine into a 15 ml labeled sterile culture tube

  • Supplement the medium with the following:

Mazen Zaharna Molecular Biology 1/2009


2 cell culture2

2- Cell Culture

Incubation

  • Add 1 ml of whole heparinized blood into the tube containing the supplemented medium

  • Mix contents of tube with gentle inversion

  • Incubate in 5% CO2 incubator at 37oC for 72 hours

Mazen Zaharna Molecular Biology 1/2009


3 stopping cell division at metaphase

3- Stopping cell division at Metaphase

  • Pre-warm the Colchicine (0.04 mg/ml) in incubator at 37oC

  • Add 25 µl of pre-warmed Colchicine to the culture

  • Mix gently and incubate at 37oC for 30-60 minutes

Mazen Zaharna Molecular Biology 1/2009


4 hypotonic treatment of red white blood cells

4- Hypotonic treatment of red & white blood cells

  • Centrifuge for 10 minutes at 2000 rpm

  • Discard supernatant without disturbing the cells leaving 0.5 ml of fluid

  • Add 1 ml of pre-warmed hypotonic solution (0.075 M KCl) at 37oC

  • Mix and then add 9 ml of hypotonic solution

  • Mix well by Pasteur pipette

  • Incubate at 37oC incubator for 17 minutes

  • hypotonic solution should not be in contact with cells more than 27 minutes (may cause rupture of WBCs)

Mazen Zaharna Molecular Biology 1/2009


5 fixation

5- Fixation

  • Fixative must be prepared fresh

  • Add 3 parts of chilled absolute methanol: 1 part glacial acetic acid

Mazen Zaharna Molecular Biology 1/2009


5 fixation1

5- Fixation

  • Centrifuge for 10 minutes at 1000 – 1500 rpm

  • Remove supernatant leaving about 0.5 ml of fluid on top of cells

  • At this time there is probably a small whitish or reddish film at the bottom of the tube

  • The film contain red blood cell debris and enlarged WBCs

Mazen Zaharna Molecular Biology 1/2009


5 fixation2

5- Fixation

  • Add 5 ml of fixative to the tube

  • Mix with a Pasteur pipette 3-4 times

  • Place in refrigerator for 30 minutes

  • Centrifuge the tube for 10 minutes at 1000-1500 rpm

  • Remove supernatant and add another 6 ml of cold fixative, & mix well

  • Centrifuge the tube for 10 minutes at 1000-1500 rpm

  • Repeat the last two steps

  • Remove the supernatant leaving 1 ml of fluid at the bottom

  • The remaining material will be used to make the slides

Mazen Zaharna Molecular Biology 1/2009


6 slides preparation

6- Slides Preparation

  • The slide must be exceptionally clean

  • Lay slides on a paper towel

  • Withdraw a few drops of cell suspension into a pipette

  • From a height of 20 cm, drop 2 or 3 drops of fluid on each slide

  • Allow the slides to dry

Mazen Zaharna Molecular Biology 1/2009


7 staining

7- Staining

  • Stain the slides by immersion in fresh Giemsa stain for 7-10 minutes

  • Remove slides from stain & rinse in distilled water

  • Observe under microscope X40 then under oil immersion

Mazen Zaharna Molecular Biology 1/2009


Human metaphase chromosomes

Mazen Zaharna Molecular Biology 1/2009


Human metaphase chromosomes

Mazen Zaharna Molecular Biology 1/2009


Human metaphase chromosomes

  • http://www.biology.arizona.edu/human_bio/activities/karyotyping/patient_a/patient_a.html

  • http://www.youtube.com/watch?v=E0WkZr819UU

Mazen Zaharna Molecular Biology 1/2009


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