Human Chromosomes Identification by GBanding Karyotyping MOLECULAR BIOLOGY Experiment Objectives Preparing Staining and Observing Gbanding human chromosomes Develop an understanding of karyotyping and the association of various chromosomal abnormalities to diseases ID: 910719
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Slide1
Mazen Zaharna Molecular Biology 1/2009
Human Chromosomes Identification by G-Banding
Karyotyping
Slide2MOLECULAR BIOLOGY
Experiment Objectives
Preparing, Staining and Observing G-banding human chromosomes
Develop an understanding of karyotyping and the association of various chromosomal abnormalities to diseases.
Slide3Human 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)
MOLECULAR BIOLOGY
Slide4Chromosome abnormalities
Chromosome abnormalities can benumerical, as in the presence of
extra
or missing chromosomes,
or structural as in translocations, inversions, large scale deletions or duplications.
MOLECULAR BIOLOGY
Slide5Chromosomal abnormalities that can be detected by karyotyping
Philadelphia Chromosome - CML
MOLECULAR BIOLOGY
Slide6cri-du-chat syndrome
Chromosome 5p deletion in cri-du-chat syndrome
Mazen Zaharna Molecular Biology 1/2009
Slide7Chromosomal 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)
MOLECULAR BIOLOGY
Slide8Chromosomal abnormalities that can be detected by karyotyping
MOLECULAR BIOLOGY
Slide9Why 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.
MOLECULAR BIOLOGY
Slide10Situations where analysis is strongly recommended
Problems with early growth & development
Fertility problems
Neoplasia
Pregnancy in older women
MOLECULAR BIOLOGY
Slide11What 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)
MOLECULAR BIOLOGY
Slide12Mazen Zaharna Molecular Biology 1/2009
21 22 x y
Slide13How 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
MOLECULAR BIOLOGY
Slide14Mazen Zaharna Molecular Biology 1/2009
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.
Slide15Performing 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.
MOLECULAR BIOLOGY
Slide16Chromosome banding
Chromosomes are stained with various dyes enabling the chromosome segments to be identifiedMost methods can distinguish 550 bands/ haploid setHigh resolution methods can distinguish up to 850 bands/ haploid set that can allow identification of small interstitial deletions
MOLECULAR BIOLOGY
Slide17G-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.
MOLECULAR BIOLOGY
Slide18G-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.
MOLECULAR BIOLOGY
Slide19Chromosome 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.
MOLECULAR BIOLOGY
Slide20Overview of Procedure
Collection of bloodCell culture
Stopping the cell division at Metaphase
Hypotonic treatment of red & white blood cells
Fixation
Slide preparation
MOLECULAR BIOLOGY
Slide21Overview of Procedure
Slide dehydrationTreatment with enzyme
Staining
MOLECULAR BIOLOGY
Slide22Monitor 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
MOLECULAR BIOLOGY
Slide237- Slide dehydration
Place fixed, dry slides on slide rack in 60
o
C oven
Bake for 3 days
Allow to cool before proceeding to the next step
MOLECULAR BIOLOGY
Slide248- Treatment with enzyme
Prepare 0.025% trypsin solution fresh, by mixing 5 ml of 0.25% trypsin with 45 ml Hank’s solutionImmerse slide in 0.025 % trypsin for 10-120 secondsRemove slide from trypsin and immediately immerse in phosphate buffer to stop trypsin action
MOLECULAR BIOLOGY
Slide25Determination 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
15
3.0
3-20 days
30
3.5
20+ days
45
4.0
Cell Concentration
< 20 mitosis
15
3.0
20-50 mitosis
30
3.5
50+ mitosis
45
4.5
MOLECULAR BIOLOGY
Slide269- Staining
Prepare a dilution of Giemsa stain by mixing 1 part of Giemsa stain with 3 parts of Phosphate bufferFlood slide with Giemsa stain for 2 minutesRinse slides thoroughly with distilled water
Allow slides to drain, then place on 60
o
C slide warming tray until completely dry
MOLECULAR BIOLOGY
Slide27Mazen Zaharna Molecular Biology 1/2009
Slide28Mazen Zaharna Molecular Biology 1/2009
Slide29Mazen Zaharna Molecular Biology 1/2009
Slide30DOWN SYNDROME
Mazen Zaharna Molecular Biology 1/2009
Slide31DOWN SYNDROME
Mazen Zaharna Molecular Biology 1/2009
Slide32TURNER SYNDROM
Mazen Zaharna Molecular Biology 1/2009
Slide33DELETION IN X
Mazen Zaharna Molecular Biology 1/2009
Slide34DEL IN X
Mazen Zaharna Molecular Biology 1/2009
Slide35RING X
Mazen Zaharna Molecular Biology 1/2009
Slide36Robertsonian translocation
IN 14
Mazen Zaharna Molecular Biology 1/2009
Slide37KLINFILTER SYNDROME
Mazen Zaharna Molecular Biology 1/2009
Slide38Triple X syndrome
Mazen Zaharna Molecular Biology 1/2009
Slide39DOUBLE YY SYNDROME
Mazen Zaharna Molecular Biology 1/2009
Slide40Edwards syndromeTRISOMY 18
Mazen Zaharna Molecular Biology 1/2009
Slide41Patau Syndrome
TRISOMY 13
Mazen Zaharna Molecular Biology 1/2009