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KARYOTYPE
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Karyotype:
A karyotype is the number and appearance ofchromosomes in the nucleus of a eukaryotic cell.
The term is also used for the complete set of
chromosomes in a species or an individual organism
Karyotypes describe the number of chromosomes,
and what they look like under a light microscope
Attention is paid to their length, the position of the
centromeres, banding pattern, any differencesbetween the sex chromosomes, and any other
physical characteristics
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Idiogram:
An ideogram or ideograph (from Gk. idea "idea"+ grafo "to write") is a graphic symbol that
represents an idea or concept When the chromosomes are depicted (by rearranging a
microphotograph) in a standard format - is known as a
karyogram or ideogram/idiogram - in pairs, ordered by
size and position of centromere for chromosomes ofthe same size
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Historical:
Prior to 1950: sections of testis (from dead humans)
1952: T. C. Hsu employed skin and spleen from 4month old fetus..good chromosome spreads
It was found after this article had been sent to pressthat the well spread metaphases and mitoticanaphases were the result of an accident. Instead of
being washed in isotonic saline, the cultures had
been washed in hypotonic Tyrode solution before
fixation
The diploid human chromosome number was reported
as 48!!!
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1956:
Tjio and Levan - used connective tissue cells fromlungs of legally aborted human embryo
Cultured in bovine amniotic fluid
Added colchicine 12hrs. prior to making chromosome
preparations
Employed Hsus hypotonic treatment to get spreads
We do not wish to generalize our present findings into
a statement that the chromosome number of man is2n=46, but it is hard to avoid the conclusion that this
would be the most natural explanation of our
observations
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Two further developments:
Ford & Harnden obtained cells from human bone
marrow and skin biopsies..showed the possibility ofmaking chromosome preparations from live adults!
Hungerford Human WBC can be stimulated to cometo mitosis by using Phytohaemagglutinin
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Karyotypes are used to study chromosomalaberrations
Used to determine other macroscopically visible
aspects of an individual's genotype, such as sex
To see the chromosomes and determine their size and
internal pattern, they are chemically labelled with a
dye ("stained")
The pattern of individual chromosomes is calledchromosome banding
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Karyotyping can be done using a sample of blood, bone
marrow, amniotic fluid, or placental tissue.
Possible due to cell culture techniques
Bone marrow dividing mitotic cells
Amniotic fluid contains fetal nucleated cells
Chorionic villi mixture of maternal and fetal cells
Human WBC
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Cells separated out from blood/amniotic fluid by
centrifugation
Stimulated to divide by chemical/hormonal treatment
Grown in special culture media
Cells treated with colchicine/colcemid
Hypotonic solution
Air dry preparation
Staining spread cells
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Cell culture:
100ml McCoys 5A medium
12.5ml fetal calf serum
2ml L-glutamine
2.5ml reconstituted PHA
2ml penicillin/streptomycin/gentamycin sulfate
To 9.5ml of this mixture
0.4ml of whole blood/Heparinized whole blood
Incubated at 37C
10 g/ml Colcemid G incubate for 1-1.5 hrs prior toharvesting cells
Total incubation time 70-72 hrs.
Cells quickly harvested for air dry preparations
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Metaphase spreads are drawn using camera lucida or
photographed
Homologous chromosomes can be recognized by size,
shape (caliper) and/or banding pattern (G, C, R, Q)
The photographed chromosomes are cut out,
matched with its partner and arranged from largestto smallest on a chart
Human idiogram consists of 7 groups:
A: 1-3 E: 16-18
B: 4, 5 F: 19, 20
C: 610, 11, 12, X G: 21, 22, Y
D: 13-15
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Normal karyotypes for women contain two Xchromosomes and are denoted 46,XX;
Men have both an X and a Y chromosome
denoted 46,XY.
However, some individuals have other karyotypes
with added or missing sex chromosomes
47,XYY, 47,XXY, 47,XXX and 45,X.
The karyotype 45,Y does not occur, as an embryo
without an X chromosome cannot survive
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GTL-Banding:
GTL-Banding /G-Banding - Giemsa/Trypsin/Leishman
banding.
Devised by Dr. Giemsa uses trypsin to partially digest the
histones the chromosome relax, letting the Leishmanstain bind to the exposed DNA
Preferentially stains the regions of DNA - rich in AT
The resulting bands are specific to each chromosome andthe order and size of the band enables us to distinguish
and compare homologous pairs of chromosomes
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G-Banding
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G-Banded Karyotype
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1971: at a conference in Paris, scientists got together to draw up a system of
numbering of the bands and sub bands.
The result is the Paris Conference ideogram.. In making the assignments,however, they did make one mistake.
Chromosome 22 has more DNA than chromosome 21 and thus the numbers
should have been reversed. Since an extra chromosome 21 was already
associated with Down syndrome, it was decided not to change the numbering
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Q-banding: Quinacrine gives bands that fluoresce on exposure to UV
light. The patterns can be correlated with G-bands.
Disadvantage: Intensity of fluorescence fades rapidly. Photographs
must be made within a few minutes of staining.
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R-banding:
It is the reverse pattern of G bands so that G-
positive bands are light with R-banding methods,
and vice versa. Involves pretreating cells with a hot salt solution
that denatures DNA that is rich in adenine and
thymine.
The chromosomes are then stained with Giemsa
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Helpful for analyzing the structure of chromosome ends,
since these areas usually stain light with G-banding
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C-banding: stains areas of heterochromatin, which
is tightly packed and repetitive DNA
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NOR-staining: refers to a silver staining method.
Identifies genes for ribosomal RNA that were active in
a previous cell cycle
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6 characteristics of karyotypes are usually observed and compared:
1. Differences in absolute sizes of chromosomes. Chromosomes can vary in
absolute size by as much as twenty-fold between genera of the same family -
Probably reflects different amounts of DNA duplication.
2. Differences in the position of centromeres - brought about by
inversions/translocations
3. Differences in relative size of chromosomes - can only be caused by segmental
interchange of unequal lengths.4. Differences in basic number of chromosomes - may occur due to successive
unequal translocations which finally remove all the essential genetic material
from a chromosome, permitting its loss without penalty to the organism
(dislocation hypothesis)
Humans have one pair fewer chromosomes than the great apes, but thegenes have been mostly translocated (added) to other chromosomes
5. Differences in number and position of satellites
6. Differences in degree and distribution ofheterochromatic regions
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Implications:
Chromosomal types and number
Numerical variations Aneuploids/Ploids Structural variations
Genetic mosaics
Phylogenetic relationships between species