Human Metaphase Chromosomes Experiment Objectives Preparing staining and
Human Metaphase Chromosomes
Experiment Objectives • Preparing, staining and observing human metaphase chromosomes. Molecular Biology
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 Molecular Biology
Cell division • Cell division can be divided into: q Interphase, q Mitosis q Prophase, q Metaphase, q Anaphase, q Telophase. q Cytokinesis Molecular Biology
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. Molecular Biology
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. Molecular Biology
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 Molecular Biology
Mazen Zaharna Molecular Biology 1/2009
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 Molecular Biology
Overview of Procedure 1. 2. 3. 4. 5. 6. 7. Collection of blood Cell culture Stopping the cell division at Metaphase Hypotonic treatment of red & white blood cells Fixation Slide preparation Staining Molecular Biology
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). Molecular Biology
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, Molecular Biology
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: Penicillin-Streptomycin Stock solution 10 µl (100000 u penicillin/ml – 100 mg/ml Streptomycin Phytohemagglutinin 0. 3 ml 20 µg/ml Fetal bovine Serum 20% 2 ml Molecular Biology
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 o • Incubate in 5% CO 2 incubator at 37 C for 72 hours Molecular Biology
3 - Stopping cell division at Metaphase • Pre-warm the Colchine (0. 04 mg/ml) in incubator at 37 o. C • Add 25 µl of pre-warmed Colchine to the culture o • Mix gently and incubate at 37 C for 30 -60 minutes Molecular Biology
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 37 o. C • Mix and then add 9 ml of hypotonic solution • Mix well by Pasteur pipette • Incubate at 37 o. C incubator for 17 minutes • hypotonic solution should not be in contact with cells more than 27 minutes (may cause rupture of WBCs) Molecular Biology
5 - Fixation • Fixative must be prepared fresh • Add 3 parts of chilled absolute methanol: 1 part glacial acetic acid Molecular Biology
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 Molecular Biology
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 Molecular Biology
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 Molecular Biology
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 X 40 then under oil immersion Molecular Biology
Mazen Zaharna Molecular Biology 1/2009
Mazen Zaharna Molecular Biology 1/2009
• http: //www. biology. arizona. edu/human_bio /activities/karyotyping/patient_a. html • http: //www. youtube. com/watch? v=E 0 Wk. Zr 819 UU Molecular Biology
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