Lab # 4&5 Human karyotype

General GeneticsGeneral Genetics

1. Students will be able to demonstrate a microtechnique for reliable chromosomal analysis of leucocytes obtained from blood.

2. Students will be able to prepare a karyotype from the chromosomes of a normal human male or female.

3. Students will be able to use the karyotyping techniques for diagnosing a chromosomal disorder.

Chromosomal aberrations are abnormalities in the number or microscopically observable structure of chromosomes.

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.

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 7 groups (A, B, C, D, E, F, G).

Group A contains pairs #1, #2, and #3 chromosomes .

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 Down’s syndrome syndrome and Klinefelter’s syndrome 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 arranged, 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.

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 red cells and remove some chromosomes protein.

After staining by Giemsa Staine, chromosomes can be microscopically observed and evaluated for abnormalities.

Heparinzed whole bloodHeparinzed whole blood Heparin sodium injectionHeparin sodium injection Peripheral blood Karyotyping medium with PHA Peripheral blood Karyotyping medium with PHA

(RPMI 1640)(RPMI 1640) Incubator 5%CO2 at 37ºCIncubator 5%CO2 at 37ºC Colcemide solution 10µg/mlColcemide solution 10µg/ml 0.075 M kcl0.075 M kcl Fixative solution ( 3x methanol : 1x glacial Fixative solution ( 3x methanol : 1x glacial

acetic acid )acetic acid ) Giemsa stain solutionGiemsa stain solution Slides and MicroscopeSlides and Microscope

1. Inoculate 0.5ml of heparinized whole blood into tube with 10ml of karyotyping medium .

2. Incubate the tubes in incubator with 5% CO2 at 37ºC for total of 72 hours.

3. After total of 69 hours from seeding add 100μl of Colcemid Solution to each culture tubes.

4. Incubate the tubes at 37oC for an additional 20-30 minutes.

5. Spin at 500g (1500 rpm) for 7 minutes.

6. Remove the supernatant and re-suspend the cells in 5ml of hypotonic 0.075M KCl pre wormed to 37ºC.

7. Incubate at 37oC for 15 minutes.8. Add drop-by- drop (with vortexing) 1ml fresh ice

cold fixative. 9. Spin at 500g (1500RPM) for 7 minutes.10. Remove the supernatant, agitate the cellular

sediment and add drop-by- drop (with continous vortexing), 5ml 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 500g (1500RPM) for 7 minutes.

14. Re-suspend the cell pellet with a 1.5ml of fresh fixative. 15. Drop 4-5 drops, from ahigh of approximately 30cm onto

aclean 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 100x and photograph it and cut each chromosome from the photograph and arrange the chromosomes according to the size and position of centomer.