2. Mazen Zaharna Molecular Biology 1/2009 Human Chromosomes Identification by G-Banding Karyotyping

3. MOLECULAR BIOLOGY Experiment Objectives Preparing, Staining and Observing G- banding human chromosomes Develop an understanding of karyotyping and the association of various chromosomal abnormalities to diseases.

4. Human Chromosomes A “normal” human carries 23 PAIRS of chromosomes (1 set came from the mother, 1 set came from the father) –22 of these sets are called autosomes (or “self chromosomes”) –1 set are the sex chromosomes A female carries two X chromosomes (XX) A male carries an X chromosome and a Y chromosome (XY) MOLECULAR BIOLOGY

5. Why do scientists look at chromosomes? Scientists can diagnose or predict genetic disorders by looking at chromosomes. This kind of analysis is used in prenatal testing and in diagnosing certain disorders, such as – Down syndrome, –or in diagnosing a specific types of leukemia. MOLECULAR BIOLOGY

6. Chromosome abnormalities Chromosome abnormalities can be –numerical, as in the presence of extra or missing chromosomes, –or structural as in translocations, inversions, large scale deletions or duplications. MOLECULAR BIOLOGY

7. Situations where analysis is strongly recommended  Problems with early growth & development  Fertility problems  Neoplasia  Pregnancy in older women MOLECULAR BIOLOGY

8. What is a Karyotype?  A display or photomicrograph of an individual’s somatic-cell metaphase chromosomes that are arranged in a standard sequence (usually based on number, size, and type) MOLECULAR BIOLOGY

9. Performing a Karyotype The slides are scanned for metaphase spreads and usually 10 to 30 cells are analyzed under the microscope by a cytogeneticist. When a good spread (minimum number of overlapping chromosomes) is found, a photograph is taken or the analysis is done by a computer. The chromosomes are arranged in a standard presentation format of longest to shortest. MOLECULAR BIOLOGY

10. How Do Scientists Identify Chromosomes? Three key features to identify their similarities and differences:  Size. This is the easiest way to tell two different chromosomes apart.  Banding pattern. The size and location of Giemsa bands on chromosomes make each chromosome pair unique.  Centromere position. Centromeres are regions in chromosomes that appear as a constriction. Using these key features, scientists match up the 23 pairs MOLECULAR BIOLOGY

11. Mazen Zaharna Molecular Biology 1/2009 In metacentric chromosomes, the centromere lies near the center of the chromosome. Submetacentric & very Submetacentric chromosomes, have a centromere that is off-center, so that one chromosome arm is longer than the other. In acrocentric chromosomes, the centromere resides very near one end.

12. Chromosome banding Chromosomes are stained with various dyes enabling the chromosome segments to be identified Most methods can distinguish 550 bands/ haploid set High resolution methods can distinguish up to 850 bands/ haploid set that can allow identification of small interstitial deletions MOLECULAR BIOLOGY

13. G-Banding Dye gives chromosomes a striped appearance because it stains the regions of DNA that are rich in adenine (A) and thymine (T) base pairs. MOLECULAR BIOLOGY

14. G-Banding Regions that stain as dark G bands replicate late in S phase of the cell cycle and contain more condensed chromatin, While light G bands generally replicate early in S phase, and have less condensed chromatin. MOLECULAR BIOLOGY

15. Chromosome Groups GroupChromosomesDescription A1–3Largest; 1 and 3 are metacentric but 2 is submetacentric B4,5Large; submetacentric with two arms very different in size C6–12,XMedium size; submetacentric D13–15Medium size; acrocentric with satellites E16–18Small; 16 is metacentric but 17 and 18 are submetacentric F19,20Small; metacentric G21,22,YSmall; acrocentric, with satellites on 21 and 22 but not on the Y Autosomes are numbered from largest to smallest, except that chromosome 21 is smaller than chromosome 22. MOLECULAR BIOLOGY

16. Chromosomal Abnormalities Alterations in chromosome number. –Euploid - normal set (2n) –Polyploidy – extra set of the entire genome. (3n, 4n etc) –Aneuploidy – the number of chromosomes is not a multiple of the normal haploid number. Monosomy –one member of a chromosome pair is missing, (2n-1) Trisomy –one chromosome set consists of 3 copies of a chromosome, (2n+1) MOLECULAR BIOLOGY

17. Chromosomal abnormalities that can be detected by karyotyping MOLECULAR BIOLOGY

18. Chromosomal abnormalities that can be detected by karyotyping Philadelphia Chromosome - CML MOLECULAR BIOLOGY

19. Overview of Procedure 1.Collection of blood 2.Cell culture 3.Stopping the cell division at Metaphase 4.Hypotonic treatment of red & white blood cells 5.Fixation 6.Slide preparation MOLECULAR BIOLOGY

20. Overview of Procedure 7.Slide dehydration 8.Treatment with enzyme 9.Staining MOLECULAR BIOLOGY

21. Monitor the quality of chromosome spreading Monitor the quality of chromosome spreading under phase contrast. Chromosomes should be well spread –without visible cytoplasm, –should appear dark grey under phase contrast MOLECULAR BIOLOGY

22. 7- Slide dehydration Place fixed, dry slides on slide rack in 60 o C oven Bake for 3 days Allow to cool before proceeding to the next step MOLECULAR BIOLOGY

23. 8- Treatment with enzyme Prepare 0.025% trypsin solution fresh, by mixing 5 ml of 0.25% trypsin with 45 ml Hank’s solution Immerse slide in 0.025 % trypsin for 10- 120 seconds Remove slide from trypsin and immediately immerse in phosphate buffer to stop trypsin action MOLECULAR BIOLOGY

24. Determination of Trypsin and Staining time Trypsin Time (seconds)Staining Time (minutes) Cell Source Lymphoblastoid304.0 Blood Lymphocytes153.0 Age of Oven Dried Slides 0-3 days153.0 3-20 days303.5 20+ days454.0 Cell Concentration < 20 mitosis153.0 20-50 mitosis303.5 50+ mitosis454.5 MOLECULAR BIOLOGY

25. 9- Staining Prepare a dilution of Giemsa stain by mixing 1 part of Giemsa stain with 3 parts of Phosphate buffer Flood slide with Giemsa stain for 2 minutes Rinse slides thoroughly with distilled water Allow slides to drain, then place on 60 o C slide warming tray until completely dry MOLECULAR BIOLOGY

26. Mazen Zaharna Molecular Biology 1/2009 21 22 x y

27. Mazen Zaharna Molecular Biology 1/2009