This document describes the process of karyotyping and identifying human chromosomes through G-banding. Key points include: - Karyotyping involves preparing, staining, and observing human chromosomes to diagnose genetic disorders and identify chromosomal abnormalities. - Humans normally have 23 pairs of chromosomes, including 22 autosomes and 1 sex chromosome. G-banding allows identification of chromosomes based on size, banding pattern, and centromere position. - The karyotyping process involves culturing cells, arresting cell division, staining chromosomes, and analyzing spreads under a microscope to identify numerical or structural abnormalities. G-banding produces dark and light bands that make each chromosome pair unique. - Common chrom

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

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

17. Chromosomal abnormalities that can
be detected by karyotyping
Mazen Zaharna Molecular
Philadelphia Chromosome - CML
Biology 1/2009

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. Overview of Procedure
7. Slide dehydration
8. Treatment with enzyme
9. Staining
Mazen Zaharna Molecular
Biology 1/2009

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

26. Mazen Zaharna Molecular
Biology 1/2009

27. Mazen Zaharna Molecular
Biology 1/2009

28. Mazen Zaharna Molecular
Biology 1/2009