1. Human Chromosomes Identification by G-Banding
Karyotyping
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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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3. Mazen Zaharna Molecular Biology 1/2009
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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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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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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6. 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).
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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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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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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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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.
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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
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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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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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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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16. Chromosomal abnormalities that can be detected by karyotyping
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17. Chromosomal abnormalities that can be detected by karyotyping
Philadelphia Chromosome - CML
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Overview of Procedure
Collection of blood
Cell culture
Stopping the cell division at Metaphase
Hypotonic treatment of red & white blood cells
Fixation
Slide preparation
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Overview of Procedure
Slide dehydration
Treatment with enzyme
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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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
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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 % trypsin for 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)
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
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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
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x y
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