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
2. Experiment Objectives
• Preparing, Staining and Observing Gbanding human chromosomes
• Develop an understanding of karyotyping
and the association of various
chromosomal abnormalities to diseases.
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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)
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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.
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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.
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Biology 1/2009
6. Situations where analysis is
strongly recommended
Problems with early growth &
development
Fertility problems
Neoplasia
Pregnancy in older women
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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)
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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.
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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
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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
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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.
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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.
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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.
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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)
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17. Chromosomal abnormalities that can
be detected by karyotyping
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Philadelphia Chromosome - CML
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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
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19. Overview of Procedure
7. Slide dehydration
8. Treatment with enzyme
9. Staining
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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
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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
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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
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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
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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
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Biology 1/2009
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 10-13 weeks after the last period, earlier than amniocentesis (which is carried out at 15-18 weeks).