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Human Chromosomes
Identification by G-Banding

Karyotyping
Mazen Zaharna Molecular
Biology 1/2009
Experiment Objectives
• Preparing, Staining and Observing Gbanding human chromosomes
• Develop an understanding of karyoty...
Human Chromosomes
• A “normal” human carries 23 PAIRS of
chromosomes (1 set came from the
mother, 1 set came from the fath...
Why do scientists look at
chromosomes?
• Scientists can diagnose or predict genetic
disorders by looking at chromosomes.
•...
Chromosome abnormalities
• Chromosome abnormalities can be
– numerical, as in the presence of
• extra
• or missing chromos...
Situations where analysis is
strongly recommended
 Problems with early growth &
development
 Fertility problems
 Neopla...
What is a Karyotype?
 A display or photomicrograph of
an individual’s somatic-cell
metaphase chromosomes that
are arrange...
Performing a Karyotype
• The slides are scanned for metaphase spreads
and usually 10 to 30 cells are analyzed under
the mi...
How Do Scientists Identify Chromosomes?
•

•

Three key features to identify their
similarities and differences:
 Size. T...
In metacentric chromosomes, the centromere lies near the
center of the chromosome.
Submetacentric & very Submetacentric ch...
Chromosome banding
• Chromosomes are stained with various
dyes enabling the chromosome segments
to be identified
• Most me...
G-Banding
Dye gives chromosomes a striped appearance
because it stains the regions of DNA that are rich in
adenine (A) and...
G-Banding
• Regions that stain as dark G bands
replicate late in S phase of the cell cycle
and contain more condensed chro...
Chromosome Groups
Group Chromosomes

Description

A

1–3

Largest; 1 and 3 are metacentric but 2 is submetacentric

B

4,5...
Chromosomal Abnormalities
•

Alterations in chromosome number.
– Euploid - normal set (2n)
– Polyploidy – extra set of the...
Chromosomal abnormalities that can
be detected by karyotyping

Mazen Zaharna Molecular
Biology 1/2009
Chromosomal abnormalities that can
be detected by karyotyping

Mazen Zaharna Molecular
Philadelphia Chromosome - CML
Biolo...
Overview of Procedure
1.
2.
3.
4.

Collection of blood
Cell culture
Stopping the cell division at Metaphase
Hypotonic trea...
Overview of Procedure
7. Slide dehydration
8. Treatment with enzyme
9. Staining

Mazen Zaharna Molecular
Biology 1/2009
Monitor the quality of chromosome
spreading
• Monitor the quality of chromosome
spreading under phase contrast.
• Chromoso...
7- Slide dehydration
• Place fixed, dry slides on slide rack in 60 oC
oven
• Bake for 3 days
• Allow to cool before procee...
8- Treatment with enzyme
• Prepare 0.025% trypsin solution fresh, by
mixing 5 ml of 0.25% trypsin with 45 ml
Hank’s soluti...
Determination of Trypsin and
Staining time
Trypsin Time (seconds)

Staining Time (minutes)

Lymphoblastoid

30

4.0

Blood...
9- Staining
• Prepare a dilution of Giemsa stain by
mixing 1 part of Giemsa stain with 3 parts
of Phosphate buffer
• Flood...
Mazen Zaharna Molecular
21
Biology 1/2009

22

x

y
Mazen Zaharna Molecular
Biology 1/2009
Mazen Zaharna Molecular
Biology 1/2009
Mazen Zaharna Molecular
Biology 1/2009
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Exp. 4 karyotyping

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Exp. 4 karyotyping

  1. 1. Human Chromosomes Identification by G-Banding Karyotyping Mazen Zaharna Molecular Biology 1/2009
  2. 2. Experiment Objectives • Preparing, Staining and Observing Gbanding human chromosomes • Develop an understanding of karyotyping and the association of various chromosomal abnormalities to diseases. Mazen Zaharna Molecular Biology 1/2009
  3. 3. 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) Mazen Zaharna Molecular Biology 1/2009
  4. 4. 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. Mazen Zaharna Molecular Biology 1/2009
  5. 5. 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. Mazen Zaharna Molecular Biology 1/2009
  6. 6. Situations where analysis is strongly recommended  Problems with early growth & development  Fertility problems  Neoplasia  Pregnancy in older women Mazen Zaharna Molecular Biology 1/2009
  7. 7. 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) Mazen Zaharna Molecular Biology 1/2009
  8. 8. 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. Mazen Zaharna Molecular Biology 1/2009
  9. 9. 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 Mazen Zaharna Molecular Biology 1/2009
  10. 10. 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. Mazen Zaharna Molecular Biology 1/2009
  11. 11. 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 Mazen Zaharna Molecular Biology 1/2009
  12. 12. 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. Mazen Zaharna Molecular Biology 1/2009
  13. 13. 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. Mazen Zaharna Molecular Biology 1/2009
  14. 14. 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. Mazen Zaharna Molecular Biology 1/2009
  15. 15. 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) Mazen Zaharna Molecular Biology 1/2009
  16. 16. Chromosomal abnormalities that can be detected by karyotyping Mazen Zaharna Molecular Biology 1/2009
  17. 17. Chromosomal abnormalities that can be detected by karyotyping Mazen Zaharna Molecular Philadelphia Chromosome - CML Biology 1/2009
  18. 18. Overview of Procedure 1. 2. 3. 4. Collection of blood Cell culture Stopping the cell division at Metaphase Hypotonic treatment of red & white blood cells 5. Fixation 6. Slide preparation Mazen Zaharna Molecular Biology 1/2009
  19. 19. Overview of Procedure 7. Slide dehydration 8. Treatment with enzyme 9. Staining Mazen Zaharna Molecular Biology 1/2009
  20. 20. 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 Mazen Zaharna Molecular Biology 1/2009
  21. 21. 7- Slide dehydration • Place fixed, dry slides on slide rack in 60 oC oven • Bake for 3 days • Allow to cool before proceeding to the next step Mazen Zaharna Molecular Biology 1/2009
  22. 22. 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 10120 seconds • Remove slide from trypsin and immediately immerse in phosphate buffer to stop trypsin action Mazen Zaharna Molecular Biology 1/2009
  23. 23. Determination of Trypsin and Staining time Trypsin Time (seconds) Staining Time (minutes) Lymphoblastoid 30 4.0 Blood Lymphocytes 15 3.0 0-3 days 15 3.0 3-20 days 30 3.5 20+ days 45 4.0 Previously Banded 45 4.0 < 20 mitosis 15 3.0 20-50 mitosis 30 3.5 Cell Source Age of Oven Dried Slides Cell Concentration 50+ mitosis Mazen Zaharna Molecular 45 Biology 1/2009 4.5
  24. 24. 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 oC slide warming tray until completely dry Mazen Zaharna Molecular Biology 1/2009
  25. 25. Mazen Zaharna Molecular 21 Biology 1/2009 22 x y
  26. 26. Mazen Zaharna Molecular Biology 1/2009
  27. 27. Mazen Zaharna Molecular Biology 1/2009
  28. 28. Mazen Zaharna Molecular Biology 1/2009

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