Cytogenetics
General Genetics
Dr. Attya Bhatti
Cytogenetics
Is the study of the structure and properties of chromosomes,
chromosomal
behaviour
during
mitosis
and
meiosis,
chromosomal influence on the phenotype and the factors that
cause chromosomal changes.
Related to disease status caused by abnormal chromosome
number and/or structure.
Methods for chromosomal analysis:
Karyotyping and banding
The collection of all the chromosomes is referred to as a
Karyotype.
The method used to analyze the chromosome constitution of an
individual, known as chromosome banding.
Chromosomes are displayed as a karyogram.
Obtaining and preparing cells for
chromosome analysis

Cell source:

Blood cells

Skin fibroblasts

Amniotic cells / chorionic villi
 Increasing the mitotic index
- proportion of cells in mitosis using colcemid

Synchronizing cells to analyze prometaphase chromosomes
Key procedure
In the case of peripheral (venous) blood

A sample is added to a small volume of nutrient medium containing
phytoheamagglutinin, which stimulates T lymphocytes to divide.

The cells are cultured under sterile conditions at 37C for about 3 days,
during which they divide, and colchicine is then added to each culture.

This drug has the extremely useful property of preventing formation of
the spindle, thereby arresting cell division during metaphase, the time
when the chromosomes are maximally condensed and therefore most
visible.

Hypotonic saline is then added, which causes the red blood cells to lyze
and results in spreading of the chromosomes, which are then fixed ,
mounted on a slide and stained ready for analysis
PREPARATION OF CHROMOSOMES
Karyotype Analysis
Following Steps are involved;
 Counting the number of cells, sometimes referred as
metaphase spread
 Analysis of the banding pattern of each individual chromosome
in selected cells.
 Total chr. Count is determined in 10-15 cells, but if mosaicism
is suspected then 30 or more cell count will be undertaken.
 Detailed analysis of the banding pattern of the individual
chromosomes is carried out in approx. 3-5 metaphase spread,
which shows high quality banding.
 The banding pattern of each chromosome is specific and shown
in the form of Idiogram.
MITOTIC CHROMOSOMAL SPREAD
Chromosome Banding
 Chromosome banding is developed based on the presence of
heterochromatin and euchromatin.
 Heterochromatin is darkly stained whereas euchromatin is
lightly stained during chromosome staining.
oEuchromatin, which undergoes the normal process of condensation and
decondensation in the cell cycle, and
oHeterochromatin,
which
remains
in
a
highly
throughout the cell cycle, even during interphase.
condensed
state
Euchromatin
 Exist in extended state, dispersed through the nucleus and staining diffusely.
 Early-replicating and GC rich region.
 In prokaryotes, euchromatin is the

only form of chromatin present.
Genes may oy may not expressed
Heterochromatin
darkly
1.
stained
two types
Constitutive ; always inactive an condensed.
Consist
Late
replicating and AT rich region
Present

of repetitive DNA
at identical positions on all chromosomes in all cell types of an organism.
Genes poorly expressed.
Human chromosomes 1, 9, 16, and the Y chromosome contain large regions of
constitutive heterochromatin.

Occurs
around the centromere and near telomeres.
2. Facultative;
Genetically active(decondensed) and inactive (condensed)
Variable in its expression. It varies with the cell type and may be manifested as
condensed, or heavily stained.
Types of chromosome banding
 G-banding
 C-banding
 Q-banding
 R-banding
 T-banding
Chromosomal Banding
 G-banding, gives dark bands

C-banding: C-banding stains the constitutive heterochromatin,
which usually lies near the centromere.

Q-banding: Q-banding is a fluorescent pattern obtained using
quinacrine for staining. The pattern of bands is very similar to that
seen in G-banding.

R-banding: reverse of G-banding (the R stands for "reverse").
 Dark regions are euchromatic (guanine-cytosine rich regions) and
the bright regions are heterochromatic (thymine-adenine rich
regions).

T-banding: Identifies a subset of the R bands which are
especially concentrated at the telomeres.
G-Banding
۩-
G-banding is obtained with Giemsa stain following digestion of
chromosomes with enzyme trypsin.
۩-
Giemsa stain, named after Gustav Giemsa, an early malariologist, is
used for the histopathological diagnosis of malaria and other parasites.
۩-
It is a mixture of methylene blue and eosin.
۩-
It is specific for the phosphate groups of DNA and attaches itself to
regions of DNA where there are high amounts of adenine-thymine
bonding.
۩-
it yields a series of lightly and darkly stained bands – the dark regions
tend to be heterochromatic, late-replicating and AT rich.
۩-
The light regions tend to be euchromatic, early-replicating
and GC rich .
G- Banding
Key procedure
In the case of peripheral (venous) blood

A sample is added to a small volume of nutrient medium containing
phytoheamagglutinin, which stimulates T lymphocytes to divide.

The cells are cultured under sterile conditions at 37C for about 3 days,
during which they divide, and colchicine is then added to each culture.

This drug has the extremely useful property of preventing formation of
the spindle, thereby arresting cell division during metaphase, the time
when the chromosomes are maximally condensed and therefore most
visible.

Hypotonic saline is then added, which causes the red blood cells to lyze
and results in spreading of the chromosomes, which are then fixed ,
mounted on a slide and stained ready for analysis
PREPARATION OF CHROMOSOMES
Molecular Methods for chromosomal
analysis Molecular Cytogenetics

Fluorescent in situ Hybridization (FISH)

Chromosome painting

Comparative Genomic Hybridization (CGH)

Molecular karyotyping and Multiplex
FISH(M-FISH)

Spectral Karyotyping

Array CGH
Key points
 Cytogenetic analysis usually focuses on chromosomes in
dividing cells.
 Dyes such as Quinacrine and Giemsa create banding patterns
that’s are useful in identifying individual chromosomes within
a cell.
 A karyotype shows the photographed chromosomes of a cell
arranged for cytogenetic analysis.
THANK YOU