1. Karyotyping

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

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Introduction
Chromosomes are composed of double-stranded DNA associated with specific proteins.
The nuclei of normal human somatic cells each contain 23 pairs of chromosomes.
1 set came from the mother “maternal ”, 1 set came from the father ” paternal ”.
During metaphase, chromosomes become condensed and stain intensely with basic dyes.
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4. Human Chromosomes 1 set are the sex chromosomes
22 of these sets are called autosomes (or “self chromosomes”) chromosomes, are numbered from 1 to 22 approximating decreasing size order.
1 set are the sex chromosomes
A female carries two X chromosomes (XX)
A male carries an X chromosome and a Y chromosome (XY)

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.

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

7. 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)

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Turner syndrome results from a single X chromosome (45, X or 45, X0).
Klinefelter syndrome, the most common male chromosomal disease (47, XXY)
Down syndrome, a common chromosomal disease, is caused by trisomy of chromosome 21.
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9. Chromosomal abnormalities (can be detected by karyotyping)

10. Chromosomal abnormalities (can be detected by karyotyping)
The Ph chromosome is an abnormally short chromosome 22 that is one of the two chromosomes involved in a translocation (an exchange of material) with chromosome 9: t(9;22)(q34;q11). This translocation takes place in a single bone marrow cell and, through the process of clonal expansion (the production of many cells from this one mutant cell), it gives rise to the leukemia (CML). 
ABL and BCR are normal genes on chromosomes 9 and 22, respectively. The ABL gene encodes a tyrosine kinase enzyme whose activity is tightly regulated (controlled). In the formation of the Ph translocation, two fusion genes are generated: BCR-ABL on the Ph chromosome and ABL-BCR on the chromosome 9 participating in the translocation. The BCR-ABL gene encodes a protein with deregulated (uncontrolled) tyrosine kinase activity. 
Philadelphia Chromosome - CML

11. Situations where analysis is strongly recommended
Problems with early growth & development
Fertility problems
Neoplasia
Pregnancy in older women
Chorionic villus sampling (CVS) is a form of prenatal diagnosis to determine chromosomal or genetic disorders in the fetus. It entails getting a sample of the chorionic villus ( placental tissue) and testing it. The advantage of CVS is that it can be carried out weeks after the last period, earlier than amniocentesis (which is carried out at weeks).

12. 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)

13. 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.
Actually chromosome 21 is smaller than chromosome 22.

14. 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

15. 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.

16. 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
G-Banding, C-Banding, Q-Banding, R-Banding, T-Banding
G banding - Giemsa
Q banding - Quinacrine “Used especially for Y chromosome abnormalities or mosaicism ”
R banding - Reverse “Used to identify X chromosome abnormalities”
C banding - Centromeric (heterochromatin) “Used to identify centromeres / heterochromatin”

17. 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.

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The difference between dark- and light-staining regions was believed to be caused by differences in the relative proportions of bases:
G-light bands being relatively GC-rich
G-dark bands AT-rich
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19. Chromosome Groups Group Chromosomes Description A 1–3
Largest; 1 and 3 are metacentric but 2 is submetacentric B
4,5
Large; submetacentric with two arms very different in size C
6–12,X
Medium size; submetacentric D
13–15
Medium size; acrocentric with satellites E
16–18
Small; 16 is metacentric but 17 and 18 are submetacentric F
19,20
Small; metacentric G
21,22,Y
Small; 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.

20. Overview of Procedure Collection of blood Cell culture
Stopping the cell division at Metaphase
Hypotonic treatment of red & white blood cells
Fixation
Slide preparation
Slide dehydration
Treatment with enzyme
Staining

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

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

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 % trypsin for seconds
Remove slide from trypsin and immediately immerse in phosphate buffer to stop trypsin action

24. Determination of Trypsin and Staining time
Trypsin Time (seconds)
Staining Time (minutes)
Cell Source
Lymphoblastoid 30
4.0
Blood Lymphocytes 15
3.0
Age of Oven Dried Slides
0-3 days
3-20 days
3.5
20+ days 45
Previously Banded
Cell Concentration
< 20 mitosis
20-50 mitosis
50+ mitosis
4.5

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 60oC slide warming tray until completely dry

26. x y

30. Mazen Zaharna Molecular Biology 1/2009
Mazen Zaharna Molecular Biology 1/2009

31. Thank You