Lab. 4
Human Karyotyping
Lab.
Main topics:

Background

What’s karyotype?

Chromosome Morphology

Arrangement the chromosomes

Chromosome Banding

Aberrations of Chromosomes

Karyotype importance
Background:Occasionally
chromosomal material is lost or rearranged during the
formation of gametes or during cell division of the early embryo. Such changes,
primarily the result of nondisjunction or translocation, are so severe that the pregnancy
ends in miscarriage – or fertilization does not occur at all. It is estimated that one in 156
live births have some kind of chromosomal abnormality. Some of the abnormalities
associated with chromosome structure and number can be detected by a test called a
Karyotype.
What’s karyotype? It’s can show prospective parents whether they have certain
abnormalities that could be passed on to their offspring, or it may be used to learn the
cause of a child’s disability. Karyotypes can also reveal the gender of a fetus or test for
certain defects through examination of cells from uterine fluid – a procedure called
amniocentesis – or through sampling of placental membranes. Over 400,000 karyotype
analyses are performed each year in the U.S. and Canada.
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Finally, Karyotype is:
Pictures of chromosomes cut out from a microphotograph of a cell and rearranged into
homologous
pairs
according
to
size
and
other
physical
characteristics. The
standardized arrangement of karyotypes allows researchers to discover if an individual
is a male or female and if he/she has any gross chromosomal abnormalities.
 Chromosome Morphology
Chromosome
composed
of
centromere and 2 arms. The short
chromosome arm is designated p
(petite) and the long arm q (one letter
after p).
Karyotypingis: pictures of chromosomes rearranged into homologous
pairs depends on:
1. Centromere positions
2. The length and morphology
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 Arrangement the chromosomes depends on centromere
positions:
According to the position of the centromere, the eukaryotic chromosomes may be rodshaped (telocentric and acrocentric), J-shaped (sub-metacentric) and V-shaped
(metacentric). During the cell divisions the microtubules of the spindle are get attached
with the chromosomal centromeres and move them towards the opposite poles of cell.
Beside centromere, the chromosomes may bear terminal unipolar segments called
telomeres.
A chromosome with the centromere at or near the middle is called metacentric. Asubmetacentric chromosome has a centromere somewhat displaced from the middle point.
If the centromere is obviously off center (e.g., halfway between the middle and the tip of
the chromosome), the term very sub-metacentric may be used. Acrocentric
chromosomes have their centromeres very near one end. Telocentric chromosomes,
which are absent in human cells, have their centromeres at the very tip of one end.
Certain human chromosomes may also contain a secondary constriction, which appears
as an unstained gap (also called a satellite stalk) near the tip.
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 Arrangement the chromosomes depends on the length
and morphology of chromosome:
Group
chromosomes
characteristics
A
1-3
Large metacentric
B
4 and 5
Large sub metacentric
C
6 - 12
Medium sub metacentric
D
13 - 15
Medium acrocentric with satellite
E
16 -18
Small sub metacentric
F
19 and 20
Small sub-metacentric
G
21 and 22
Small acrocentric with satellite
X+Y
 Chromosome Banding
Staining of bands, or chromosome segments, allowing the precise identification of
individual chromosomes or parts of
chromosomes. Applications include
the
determination of chromosome rearrangements in malformation syndromes and cancer,
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the chemistry of chromosome segments, chromosome changes during evolution, and,
in conjunction with cell hybridization studies, chromosome mapping.
The banding techniques fall into two principal groups: 1) those resulting in bands
distributed along the length of the whole chromosome, such as G-, Q- and R-bands and
2) those that stain a restricted number of specific bands or structures. These latter
include methods which reveal centromeric bands, C-bands, and nucleolus organizer
regions, NOR's (at terminal regions of acrocentric chromosomes). C-banding methods
do not permit identification of every chromosome in the somatic cell complement, but
can be used to identify specific chromosomes.

Q-Banding
Quinacrine mustard, an alkylating agent, was the first chemical to band chromosomes
viewed under a fluorescence microscope. Quinacrine dihydrochloride has subsequently
been substituted by quinacrine mustard. The alternating bands of bright and dull
fluorescence are called Q bands. The bright bands are primary composed of DNA rich
in adenine and thymine, while the dull bands are rich in guanine and cytosine.Q bands
are especially useful for distinguishing the human Y chromosome and various
chromosome
polymorphisms
involving
satellites
and
centromeres
of
specific
chromosomes.

G-banding
Giemsa has become the most commonly used stain in human cytogenetic analysis.
Unlike Q-banding, G-banding usually requires pre-treating chromosomes with either salt
or a proteolytic (protein-digesting) enzyme. When chromosomes are pre-treated with
the proteolytic enzyme trypsin the process is called GTG banding. Giemsa stains
preferentially regions rich in adenine and thymine. Therefore, G bands correspond
closely to Q bands.Standard G band staining techniques allow between 400 and 600
bands to be seen on metaphase chromosomes. With high resolution G-banding
techniques, as many as two thousand different bands have been catalogued on the
twenty-four human chromosomes.
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
R-banding
Reverse banding (R-banding) involves the incubation of slides containing metaphase
chromosomes in hot phosphate buffer and stained with Giemsa. The banding pattern
that results is essentially the reverse of G bands. R bands are GC-rich. The AT-rich
regions are selectively denatured by heat leaving the GC-rich regions intact.
Fluorochromes that are GC specific also produce a reverse chromosome banding
pattern. R-banding is helpful for analyzing the structure of chromosome ends, since
these areas usually stain light with G-banding.

C-Banding
C-banding stains areas of heterochromatin, which is tightly packed and repetitive DNA.
C-banding is specifically useful in humans to stain the centromeric chromosome regions
and other regions containing constitutive heterochromatin - secondary constrictions of
human chromosomes 1, 9, 16, and the distal segment of the Y chromosome long arm.

NOR-banding
NOR-banding involves silver staining (silver nitrate solution) of the "nucleolar organizing
region", which contains rRNA genes.
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Aberrations of Chromosomes:
One of the best examples of this is the translocation between human chromosomes 9
and 22 – t (9;22) (q34:q11) – or the
Dup.
Duplication
chronic myelogenous leukemia (CML).
Def.
Deficiency
For example:45, xx, -22, t (14q
Inv.
Inversion
21q).
T.
Translation
Philadelphia
chromosome
diagnostic
of
Gender: Female
No. of chromosome: 45
Aberrations: def. in Ch. 22 and T. between band 14 in q and band 21 in
q
Karyotype importance of the study:
Therefore, a full Karyotype of the object through the number, shape and type of
chromosomes and packages and add an important genetic information about this
object:
• The difference of gender is usually.
• Between sexual and physical cells.
• Among the members of the clan. (Chromosomal Polymorphism)
• Natural individuals and abnormal.
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Training
What is your explanation of some disease cases in the pictures the
following?
1. 47, XY, +X
 Gender: ..................................................
 No. of chromosome:.......................................
 Aberrations: ......................................
2. 45, X, - X
 Gender: ..................................................
 No. of chromosome:.......................................
 Aberrations: ......................................
3. 46, XY, inv (3p2, 3q2)
 Gender: ..................................................
 No. of chromosome:.......................................
 Aberrations: ......................................
4. 47, XX, +13, t (7p2, Xq2)
 Gender: ..................................................
 No. of chromosome:.......................................
 Aberrations: ......................................
References:
http://www3.nd.edu/~nismec/biomodel/mod9/9%20Human%20Karyotyping%20Activity.pdf
http://131.229.88.77/microscopy/home.html
http://www.macroevolution.net/human-karyotype-colored.html
http://arbl.cvmbs.colostate.edu/hbooks/genetics/medgen/chromo/chromosomes.html
https://www.researchgate.net/file.PostFileLoader.html?id=55849e455f7f71bd238b4582&assetKey=AS%3A273798773837829%401
442290124120
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Practice work:
The purpose of this laboratory experience is: understand what a karyotype is and
how it is performed. -understand the reason for performing a karyotype, especially for
those with a higher risk of genetic defect in their lineage. -to determine what genetic
defect is present in a chromosome sample. -to investigate a variety of genetic disorders
that commonly occur and are studied in biology classes. Materials: The following
materials are needed to perform this laboratory experience: -Scissors -tape -ruler -small
envelope.
Procedure: The following procedure is utilized to perform this laboratory experience:
1. Using the attached sheets, complete different karyotypes: normal male, normal
female, different disorders of your choice out of the four. Additional laboratory
minutes may be granted for work above and beyond the four required karyotypes.
2. Working slowly and carefully, using scissors cut out the chromosome on one page
labeled “1” and find its’ EXACT match elsewhere on the page (it will not be numbered).
Cut out this chromosome and tape BOTH chromosomes side by side on a “data page”
that has the heading filled out.
3. Continue this procedure until you have matched all chromosomes and taped each of
them in the corresponding place on the data page.
4. If you are caught short of time, use the coin envelope to store any chromosomes you
may have clipped out and not matched. Do Not Cut out All Chromosomes and Then
Attempt to Match Them!!! Cut out Only One at a Time or You Will Lose
Chromosomes.
5. in the event that you have an extra chromosome, Do Not Throw It Out! It is the
chromosome that causes your mutation/disorder and you must match it correctly.
6. Once your chromosomes are all cut out and included in the karyotypes, answer the
questions and complete the lab.
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