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Karyotyping, Chromosome Banding and

Chromosome Painting
1.Karyotyping
 Introduction
• Particular chromosome complement of an individual or a
related group of individuals, as defined by the chromosome
size, morphology and number is known as a “Karyotype”.

• Karyotype
Size of chromosome
Position of centromere
Presence of secondary constriction
Size of satellite
• Derived from Greek word “karyon”, which
means "nucleus”, karyotype is represented as
Idiogram.
• When the haploid set of chromosomes of an
organism are ordered in a series of decreasing
size, it is said to be an idiogram.
• In other sense diagrammatic
representation of a karyotype
is an Idiogram.
 History of karyotyping

• Grygorii Levitsky (1931) seems to have

been the first person to define the karyotype as
the “phenotypic appearance of
the somatic chromosomes, in contrast to
their genic contents”.
Types of Karyotype

Karyotype

Symmetric
Asymmetric
Karyotype
Karyotype
 Types of Karyotype
Asymmetric Karyotype Symmetric Karyotype
• Show larger difference • Show lesser difference
between smaller and between smaller and
larger chromosome in a larger chromosome in a
set. set.
• Have more acrocentric • Have more metacentric
chromosomes. chromosomes.
• Have relatively • Have no relatively
advanced feature advanced feature
• In 1931 G.A. Levitzky, a Russian scientist suggested that in
flowering plants there is a predominant trend towards
karyotype asymmetry. This trend has been carefully studied in
the genus Crepis of the family compositae.
 Species showing a greater asymmetry is
more advanced. HOW???
Degree of asymmetry
• Proportion of metacentric, acrocentric
chromosomes in a set.
• Ratio between size of largest and smallest
chromosomes in a set.
Interpretation
• Higher the proportion of acrocentric
chromosomes, Greater the value of size ratio,
more asymmetrical is a karyotype
Procedure of karyotyping
 Plant Cells at
Metaphase
Process of
karyotyping in
N banding
Plants technique

Root tips -
0.5 to 1cm

Pretreated with colchicine Slide preparation

for 3 hrs at 26 ᵒC
 Representation of a karyotype
• By arranging chromosomes of somatic
complement in a descending order of size
keeping their centromeres in a straight line.
Longest chromosome –on extreme left.
Shortest chromosome –on extreme right
Sex chromosomes –allosomes–extreme right
 Advantages of Karyotyping
• Reveals structural features of each
chromosomes.
• Helps in studying chromosome banding
pattern.
• Helps in the identification of chromosomal
aberrations.
• Diagnosis of prenatal genetic defects.
• Aids in studying evolutionary changes .
2.Chromosome
Banding
Introduction
History
Why to study Banding pattern??
• This allows you to see smaller pieces of the
chromosome, so that you could identify
smaller structural chromosome abnormalities
not visible on a routine analysis.
Classification of Banding Techniques

Based on
GC and AT rich regions.
Constitutive Heterochromatin Region.

Always metaphase chromosomes whose size has

condensed and whose diameter is increased are
used for chromosome banding studies after
fixing the stage.
 Banding Techniques

Q G C
N (NOR) (Centromeric)
(Quinarcine) (Giemsa)

1958 1971 1973 1978

Casperson Summer Matsui & Linde

et.al et.al Sasaki &Laursen
 1.Q Banding Techniques
Chromosome

Stained with Quinarcine Mustard

Subjected UV light

Banding Pattern

Region Rich in AT Region rich in GC

bases bases

Dark staining Light staining

AT region quenches dye & GC region quenches dye but do

fluorescence, situated in not fluorescence ,situated in
heterochromatin region euchromatin region
 Q Banding Techniques
Advantages Disadvantages
• Tendency to fade during
• Simple and Versatile. examination.
• Used where G band is  Photo-degradation .
not accepted.  Chromopore- absorb
light of a particular
• Used in study of wavelength due to a
chromosome chemical bond formed
heteromorphism. between dye and light.
 UV light breaks the
chemical bond.
 2.G Banding Techniques
Chromosome Interaction of
the DNA with
thiazine & eosin
components of
Weak Trypsin / stain brightens
urea/ protease sulphur rich
To regions
denature
protein
Treated with
Giemsa
Methylene Azure+
Methylene Violet+
Methylene Blue+
Banding pattern
Eosine
 G Banding Techniques
Advantages Disadvantages
• Used in identification of • Not used in plants.
bands rich in Sulphur
content.
• Used in the
identification of
chromosomal
abnormalities
• Gene Mapping.
 G banding not used in Plants.
Why????
• Human mitotic metaphase chromosome is 2.3
times shorter.
• Plant mitotic metaphase chromosome is 10
times more shorter than human chromosome.
• Hence difficult to demonstrate the arrangement
of bands at this level of saturation with G
banding technique.

Source: Greilhuber, J (1977). Why Plant chromosome do not

show G bands? .Theory of Applied Genetics, Vol 50(3): 121- 124.
3. N Banding Techniques
Chromosome

Air Dried

Treated with 5% Trichloroacetic

acid @ 95ᵒC for 30 min.

Treated with 0.1N HCl @ 60ᵒC for

30 min.

Banding pattern in Structural non-

histone proteins linked to NOR region
 N Banding Techniques
Advantages
• Used in the identification of Nucleolar
organizer region.
• Superior banding pattern for plants.
 4. C Banding Techniques

Chromosome DNA denaturing

Treating with alkali solution

Washing with Sodium citrate @ 60ᵒC

for 30 min. Repeatitive DNA
renature but
unique DNA do
Staining with Giemsa Solution not renature

Banding pattern at heterochromatin

region
 C Banding Techniques
Advantages
• Identification of chromosomes particularly in
insects and plants.
• Identification of bivalents at diakinesis using
both centromere position.
• Paternity testing.
• Gene mapping.
 Representation of a Chromosome Band

• Each human chromosome has a short arm ("p" for

"petit") and long arm ("q" for "queue"), separated
by a centromere.

• Each chromosome arm is divided into regions,

or cytogenetic bands, that can be seen using a
microscope and special stains.
• The cytogenetic bands are labeled p1, p2, q1,
q2, etc., counting from the centromere out
toward the telomeres.
• At higher resolutions, sub-bands can be seen
within the bands. The sub-bands are also
numbered from the centromere out toward the
telomere.
• Example 1: The cytogenetic map location of
a gene is 7q31.2.
This indicates that the gene is on chromosome
7, q arm, band 3, sub-band 1, and sub-sub-
band 2.
• The ends of the chromosomes are labeled ptel
and qtel.
• Example 2: The notation 7qtel refers to the
end of the long arm of chromosome 7.
3.Chromosome
Painting
 Introduction
• Chromosome 'painting' refers to the
hybridization of fluorescently
labeled chromosome-specific, composite
probe pools to cytological preparations.
• First termed by Pinkel et.al in 1988.
• Chromosome painting coupled with
Fluorescence in situ hybridization (FISH) is
used routinely for identification of
chromosomes.
 WHY?????
Helps in the identification of Chromosomal
rearrangements.
Helps in the identification of Chromosomal
breakpoints.
Helps in determination of extra chromosomal
material.
Procedures Sample preparation and
hybridization
• Prepare slides with metaphase chromosomes .
• Dehydrate in ethanol.
• Denature DNA at 70ᵒC.
• Denature labeled probe.
• Incubate at 37ᵒC for 4-16
hours for hybridization.
Procedure of Chromosome Painting

Target DNA
 ADVANTAGES OF FISH
Rapid
High efficiency of hybridization and
detection
Lots of cells can be analyzed
Problems with in situ hybridization

Permeabilization problems
Uneven cell penetration
 High amount of background
autofluorescene
 Conclusion
. Studies structural features of each
chromosomes.

Helps in studying Ideograms, chromosome

banding pattern and Chromosomal painting
techniques.

Helps in the identification of chromosomal

aberrations.
Helps in studying evolutionary changes.
 REFERENCE
• Gupta ,P. K., 2012. Cytogenetics an advanced
study, Chapter 1: 3-16.
• Wendy, A. B.,2001. Karyotype analysis and
chromosome banding. Nature, 1-6.
• Moore, C. M. and Best, R. G., 2002.
Chromosome preparation and banding. Nature, 1-
6.
• Ried, T., et.al ,1998. Chromosome painting : a
useful art. Human Molecular Genetics,
Vol(7),1619- 1626.
THANK YOU