1 / 24

Human Metaphase Chromosomes

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).

maximos
Download Presentation

Human Metaphase Chromosomes

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Human Metaphase Chromosomes

  2. Experiment Objectives • Preparing, staining and observing human metaphase chromosomes. Mazen Zaharna Molecular Biology 1/2009

  3. 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

  4. Cell division • Cell division can be divided into: • Interphase, • Mitosis • Prophase, • Metaphase, • Anaphase, • Telophase. • Cytokinesis Mazen Zaharna Molecular Biology 1/2009

  5. 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

  6. 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

  7. 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

  8. Mazen Zaharna Molecular Biology 1/2009

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 3- Stopping cell division at Metaphase • Pre-warm the Colchine (0.04 mg/ml) in incubator at 37oC • Add 25 µl of pre-warmed Colchine to the culture • Mix gently and incubate at 37oC for 30-60 minutes Mazen Zaharna Molecular Biology 1/2009

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. 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

  22. Mazen Zaharna Molecular Biology 1/2009

  23. Mazen Zaharna Molecular Biology 1/2009

  24. http://www.biology.arizona.edu/human_bio/activities/karyotyping/patient_a/patient_a.htmlhttp://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

More Related