KARYOTYPING

P A R T 2
Kamille Stephanie M. Samson, RMT
 The study of karyotypes is made possible by staining.
 Giemsa is applied after cells have been arrested during cell division by a solution of
colchicine.
 For humans, WBC are used most frequently because they are easily induced to divide
and grow in tissue culture.
 Sometimes observations may be made on non-dividing (interphase cells)
 The sex of an unborn fetus can be determined by observation of interphase cells
(amniotic centesis and Barr body)
 A normal human female has only one Barr body per somatic cell, while a normal
human male has none.
 A Barr body is the inactive X chromosome in a female somatic cell
Cells Used For
Chromosomal Analysis
i. Any cell with a nucleus
ii. Lymphocytes
iii. Skin cells
iv. Tumor cells
v. Amniotic cells
vi. Chorionic villi
vii. Rare fetal cells from maternal blood
  5 mL of blood is removed from the patient.
 If a fetus is being karyotyped, amniotic fluid is
removed from the amniotic sac which surrounds the
fetus during development.
 This is done with the aid of a large syringe and
ultrasound picturing.
 There are cells which have come off the fetus in this
fluid. The WBC are removed from the blood or the
living cells are removed from the amniotic fluid.
 These cells are then cultured in a medium in which
they undergo mitosis

 Mitosis is stopped at metaphase using chemicals.

KARYOTYPING
 The cells are then placed onto a slide and spread out.
They are viewed under a microscope which is
specially adapted with a camera to take a picture of
the chromosomes from one of the cells.

PROCEDURE
 Once the picture is taken and enlarged, the
chromosomes are cut out and arranged in pairs
according to size and location of the centromere
 Group A Chromosomes 1-3 are largest with median
centromere
Chromosomes are arranged into seven
groups based on size and centromere Group B Chromosome 4-5 are large with submedian
location. The centromeres can be found in centromere
the middle of the chromosome (median), Group C Chromosomes 6-12 are medium sized with
near one end (acrocentric), or in between submedian centromere
these first two(submedian) Group D Chromosomes 13-15 are medium sized with
acrocentric centromere
Group E Chromosomes 16-18 are short with median or
submedian centromere
Group F Chromosome 19-20 are short with median
centromere
Group G Chromosomes 21-22 are very short with acrocentric
centromere
Chromosome X Similar to Group C
Chromosome Y Similar to Group G
Chromosome Painting
Using Fluorescent Dyes
DNA sequences attached to fluorescent dyes
The sequences attach to the chromosome and “paint” specific regions
Using several different DNA sequences and fluorescent dyes produces
a unique pattern for each of the 24 types of human chromosomes
 6 Different i.
ii.
Differences in absolute sizes of chromosomes.
Differences in relative size of chromosomes.
Characteristics of iii.
iv.
Differences in the position of centromeres
Differences in basic number of chromosomes
Karyotypes are Usually v. Differences in number and position of satellites
vi. Differences in degree and distribution of
Observed and Compared heterochromatic regions.
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PROCEDURE:

1. De-stain slides for 10 minutes in 95%

ethanol

2. Place slide in PBS ( phosphate

Types of Banding
Buffer Solution) and incubate
for 10 MINUTES AT 56OC

3. Treat slide with 0.22 mL of 25 %

 Cytogenetics employs several techniques to visualized trypsin, 2.5 mL of methanol and
different aspects of chromosomes: 0.22 mL of stock Giemsa and 6.5
i. G-banding mL 0f PBS with a pH of 7.4.
ii. R-banding
iii. C-banding 4. Flood slide with stain solution for
iv. Q-banding
15 minutes. Rinse with water and
v. T-banding
air dry.

5. View slide under bright field, oil

immersion microscope.
 G-banding
i. G-banding is obtained with Giemsa stain
following digestion of chromosomes with
trypsin.
ii. It yields a series of lightly and darkly
stained bands
o The dark regions tend to be
heterochromatic, late-replicating
and AT rich.
o The light regions tend to be
euchromatic, early-replicating and
GC rich
iii. This method will normally produce 300-
400 bands in a normal, human genome
R-banding C-banding
i. R-banding is the reversed of G-banding i. Giemsa binds to consecutive
o The dark region are euchromatic heterochromatin, so it stains centromeres.
(guanine-cytosine rich regions)
o The bright/light regions are
heterochromatic (thymine-adenine Q-banding
rich regions) i. Q-banding is a fluorescent pattern
ii. A reverse Giemsa chromosome banding obtained using quinacrine for staining. The
method that produces bands pattern of bands is very similar to that
complementary to G-bands; induced by seen in G-banding.
treatment with high temperature, low pH, or
acridine orange staining; often used
together with G-banding on human T-banding
karyotype to determine whether there are
deletions. i. Visualize telomeres

Silver staining: Silver nitrate stains the nucleolar organization region-associated protein. This yields a dark region where the
silver is deposited, denoting the activity of rRNA genes within the NOR.
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SPECTRAL KARYOTYPING
 Spectral karyotyping is a molecular cytogenetic
technique used to simultaneously visualize all the
pairs of chromosomes in an organism in different
colors.
 Fluorescently labeled probes for each chromosome
are made by labeling chromosome-specific DNA
with different fluorophores.
 Because there are a limited number of spectrally-
distinct fluorophores, a combinatorial labeling
method is used to generate many different colors.

 Spectral differences generated by combinatorial labeling are captured and analyzed by using an
interferometer attached to a fluorescence microscope.
 Image processing software then assigns a pseudo color to each spectrally different combination, allowing the
visualization of the individually colored chromosomes.
 This technique is used to identify structural chromosome aberrations in cancer cells and other dis ease
conditions when Giemsa banding or other techniques are not accurate enough.
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DIGITAL KARYOTYPING
Digital karyotyping is a technique used to quantify the
DNA copy number on a genomic scale. Short sequences
of DNA from specific loci all over the genome are
isolated and enumerated. Thismethod is also known as
virtual karyotyping

Variation is often found:

i. Between the sexes
ii. Between the germ-line and soma
iii. Between members of a population
iv. Geographical variation between races
v. Mosaics or otherwise abnormal individuals.
 The normal human karyotypes contain 22
pairs of autosomal chromosomes and one
pair of sex chromosomes.

 Normal karyotypes for females contain two

X chromosomes and are denoted 46,XX;
males have both an X and a Y chromosome
denoted 46, XY.
 Any variation from the standard karyotype
may lead to developmental abnormalities.
CHANGES DURING DEVELOPMENT

Some organisms go in for large-scale elimination of heterochromatin,

or other kinds of visible adjustment to the karyotype.

Chromosome Chromatin
X-inactivation
Elimination Diminution
o Chromosome abnormalities can be
numerical, as in the presence of
extra or missing chromosomes, or
structural, as in derivative
chromosome, translocations, inversions,
large- scale deletions or
duplications.
o Numerical abnormalities, also known
as aneuploidy, often occur as a
result of nondisjunction during meiosis
in the formation of a gamete;
o Trisomies, in which three copies of a
chromosome a re present instead
of the usual two, are common
numerical abnormalities.
o Structural abnormalities often
arise from errors in homologous
recombination.
 Ploidy is the number of complete sets
of chromosomes in a cell.

Polyploidy Haplo-diploidy Endopolyploidy Aneuploidy

where there are more where one sex is diploid, A process by which It isthe condition in
than two sets of and the other haploid. Itis chromosomes replicate which the chromosome
homologous a common arrangement in without the division of number in the cells is not
chromosomes in the cells, the Hymenoptera, and in the cell nucleus, resulting the typical number for
occurs mainly in plants. some other groups. in a polyploid nucleus. the species.
Also called endomitosis.
Some disorders arise from loss of
just piece of one chromosome

 Cri du chat (cry of the cat), from a truncated

short arm on chromosome 5. The name comes
from the babies' distinctive cry, caused by
abnormal formation of the larynx.
 1p36 Deletion syndrome, from the loss of part
of the short arm of chromosome1.
 Angelman syndrome –50%of cases have a
segment of the long arm of chromosome 15
missing; a deletion of the maternal genes,
example of imprinting disorder.
 Prader-Willi syndrome –50%of cases have a
segment of the long arm of chromosome 15
missing; a deletion of the paternal genes,
example of imprinting disorder.
 Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual;

 One well-documented example is the Philadelphia chromosome, a translocation mutation commonly

associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia.
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