Practical Of Genetics Human karyotype Objectives 1 Students

Practical Of Genetics Human karyotype

Objectives 1. Students will be able to prepare a karyotype from the chromosomes of a normal human male or female. 2. Students will be able to use the karyotyping techniques for diagnosing a chromosomal disorder.

Introduction • The “normal” human diploid cell has 23 pairs of chromosomes visible only at the metaphase stage of mitosis, 22 homologous pairs of autosomes and two sex chromosomes. • Chromosomal aberrations are abnormalities in the number or microscopically observable structure of chromosomes.

Karyotyping • is a technique that allows for the visualization and identification of human metaphase chromosomes. • This technique can be used to assess the “normalcy” of an individual’s chromosomes and to assay for various genetic diseases such as Down’s syndrome and Klinefelter’s syndrome …etc.

• It is estimated that one in 156 live births have some kind of chromosomal abnormality. • To create a karyotype, chromosomes from a cell are stained, and photographed. • The photograph is enlarged and cut up into individual chromosomes. • The homologous chromosomes can be distinguished by length and by the position of the centromer.

• A chromosome is divided by its centromer into short arm (p) and long arm (q). chromosomes can be classified by the position of their centromer: 1. Metacentric: If its two arms are equal in length. 2. Submetacentric: If arms' lengths are unequal. 3. Acrocentric: If the p arm is so short that is hard to observe, but still present.

Chromosome Morphology Centromere Arm Short arm (p) Long arm (q) Metacentric Submetacentric Acrocentric

�Each chromosome has a characteristic size and shape in the “normal” cell. �Chromosome pair #1 contains the largest chromosomes, while the Y chromosome is the smallest. �Chromosomes of similar size and morphology are grouped together by letter; the chromosomes can be arranged in 7 groups (A, B, C, D, E, F, G).

• Group A: chromosomes 1, 2, 3 – largest – metacentric • Group B: chromosomes 4, 5 – large – submetacentric • Group C: chromosomes 6, 7, 8, 9, 10, 11, 12 – medium – submetacentric

• Group D: chromosomes 13, 14, 15 – medium – acrocentric • Group E: chromosomes 16, 17, 18 – short – metacentric or submetacentric • Group F: chromosomes 19, 20 – short – metacentric • Group G: chromosomes 21, 22 – very short – acrocentric

Visualizing Metaphase Chromosomes �A blood sample is taken and white blood cells grown in special medium for three days under the influence of the mitotic stimulant ( phytohemagglutinin " PHA " ) to enter into mitosis by DNA replication. �After 68 -72 hours, a mitotic inhibitor ( Cholchicin ) is added to the culture to stop mitosis in the metaphase stage. �After treatment by hypotonic solution ( KCl ) to causes a swelling of the cells and allow dispersion of the chromosomes within the cell membrane.

• Fixation is used as wash solution to lyse the remaining red cells and remove some chromosomes protein. • After staining by Giemsa Staine, chromosomes can be microscopically observed and evaluated for abnormalities.

Materials Required �Blood �Heparin sodium injection �Peripheral blood Karyotyping medium with PHA (RPMI 1640) �Incubator 5%CO 2 at 37ºC �Colcemide solution 10µg/ml � 0. 075 M kcl �Fixative solution ( 3 x methanol : 1 x glacial acetic acid ) �Giemsa stain solution �Slides and Microscope

Karyotyping procedure: 1. Inoculate 0. 5 ml of heparinized whole blood into tube with 10 ml of karyotyping medium. 2. Incubate the tubes in incubator with 5% CO 2 at 37ºC for total of 72 hours. 3. After total of 69 hours from seeding add Colcemid Solution to each culture tubes. 100μl of 4. Incubate the tubes at 37 o. C for an additional 20 -30 minutes. 5. Spin at (1500 rpm) for 7 minutes.

6. Remove the supernatant and re-suspend the cells in 5 ml of hypotonic 0. 075 M KCl pre wormed to 37ºC. 7. Incubate at 37 o. C for 15 minutes. 8. Add drop-by- drop (with vortexing) 1 ml fresh ice cold fixative. 9. Spin at (1500 RPM) for 7 minutes. 10. Remove the supernatant, and add drop-by- drop (with continuous vortexing), 5 ml of fresh, ice-cold fixative. 11. Leave at 4ºC for 20 minutes. 12. Repeat steps 9 and 10, until the supernatant is clear. 13. Spine at (1500 RPM) for 7 minutes.

14. Re-suspend the cell pellet with a 1. 5 ml of fresh fixative. 15. Drop 4 -5 drops, from a high of approximately 30 cm onto a clean slide and blow carefully on the drops for spreading them on the slide. 16. Put the slides on a 45ºC heated plate for 2 -4 minutes. 17. Heat the slides to 60 ºC for overnight or to 90 ºC for 90 minutes. 18. Place the slides and flood them with Giemsa stain solution for 8 minutes. 19. Gently rinse the slides in distilled water and air dry. 20. Observe the chromsomes under microscope by using 10, 40 and 100 x and photograph it and cut each chromosome from the photograph and arrange the chromosomes according to the size and position of centomer.