GENETICS

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PKDIMS
MODULE 2

KARYOTYPING
BARR BODY
KARYOTYPE
 Chromosome
constitution of an
individual
Or
 Photomicrograph of

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PKDIMS
an individual’s
chromosomes
arranged in a
standard manner
KARYOTYPING
Any tissue with living nucleated cells that
undergo division can be used for studying
human chromosomes
Specimens used for karyotyping:

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PKDIMS
 Blood – Lymphocyte culture
 Skin – Fibroblast culture
 Bone marrow
 Amniotic fluid cell culture
 Chorionic villus – direct & culture.
KARYOTYPING – PROCEDURE
SHORT TERM LYMPHOCYTE CULTURE
• Peripheral blood
added to:
• Nutrient medium
• Phytohemagglutinin
– which stimulates T
lymphocytes to
divide

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KARYOTYPING PROCEDURE - Contd..

INCUBATION :

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PKDIMS
Culture maintained
at 37 c in an
incubator
for 3 days.
KARYOTYPING PROCEDURE Contd..

• Colchicine is added to culture – prevents

formation of spindle & arrests cell division during
metaphase
• Chromosomes maximally condensed & most

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PKDIMS
easily visible in metaphase
• Hypotonic saline added – causes RBC to lyze &
results in spreading of chromosomes
KARYOTYPING PROCEDURE Contd..

• Chromosomes are fixed, mounted on a slide &

stained
• Chromosome analysis done under microscope
• Photographs taken

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• Individual chromosomes are cut & arranged
depending upon size & type
CHROMOSOME BANDING
• Staining methods to identify individual
chromosomes
• G (Giemsa) banding used most commonly
• Chromosomes are treated with trypsin

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PKDIMS
• Then stained with a DNA-binding dye – Giemsa
• Banding gives each chromosome a characteristic
& reproducible pattern of light & dark bands
KARYOTYPE – METAPHASE SPREAD

• Total number of
chromosomes counted in
a specified number of
cells
• Careful analysis of the
banding pattern of each
individual chromosome

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KARYOTYPE
• The banding pattern
of each chromosome
is specific
• Can be shown as a
stylized ideal
karyotype – idiogram
• Each pair of
homologous
chromosomes is
analyzed
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Karyotype - uses

• The sex chromosome

complement
• Any chromosomal
abnormality
• Gross structural
• Numerical

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Dr Ramakrishnan, Anatomy @
PKDIMS
 KARYOTYPE-MALE – 46, XY

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PKDIMS
 KARYOTYPE – FEMALE – 46, XX

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PKDIMS
 Chromosomes

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PKDIMS
Chromosome Nomenclature
• Each chromosome is
divided into:
• Arms
• Short arm – p

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• Long arm - q
• Region
• Bands
TYPES OF CHROMOSOMES
• Metacentric –
centromere in the
middle
• Submetacentric –

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centromere in an
intermediate position
CLASSIFICATION OF CHROMOSOMES
ACCORDING TO POSITION OF CENTROMERE
• Acrocentric –
centromere closer to
one end (acrocentric
chromosomes have

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PKDIMS
stalk-like appendages
– satellites)
• Telocentric –
centromere is at one
end
DENVER CLASSIFICATION OF
CHROMOSOMES
Classified into groups
on the basis of overall
morphology
• A = 1-3
•B=4&5
• C = 6-12 & X
• D = 13-15
• E = 16-18
• F = 19 & 20
• G = 21, 22 & Y

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FLUORESCENT IN-SITU
HYBRIDIZATION (FISH)
• Based on ability of a single
stranded DNA – ‘probe’ to
anneal with its complementary
target sequence in a
metaphase spread

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FISH
• Can be used to study
chromosomes in cells
during interphase
• Widely used for clinical
diagnostic purposes

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BARR BODY
(SEX CHROMATIN)

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PKDIMS
X INACTIVATION
• Lyonisation or Lyon’s Hypothesis

• [Link] F Lyon - 1961

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PKDIMS
• In female cells only one X is active, the other is
inactivated
• Inactivation occurs in early embryonic life (15-
16 days)
X Inactivation …
• Either of the 2 X chromosomes can be
inactivated in any particular cell
• Thereafter the same X chromosome is

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inactivated in all daughter cells
• Inactive X can be maternal or paternal X
• Randomly determined – but fixed
Mechanism of inactivation

• All normal females are mosaics for X

• 2 distinct population of cells

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• One having an active X derived from father
• One having an active X derived from mother

• Differential methylation of DNA

Sex Chromatin/Barr Body
• Inactive X
chromosome exists in
a condensed form
during interphase
• Appears as a darkly

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PKDIMS
staining mass of
chromatin
• During mitosis, the
inactive X
chromosome is late
replicating
BARR BODY
• Barr and Bertram - 1949
• Observed chromatin material in nuclei of
neurons in female cats,but not in males
• Later named as sex chromatin

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PKDIMS
Barr body..

• Cells are said to be sex

chromatin
positive/negative
depending on presence or
absence

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PKDIMS
• Present in all cells
• Can be easily
demonstrated in cells of
oral mucosa
• Number of Barr body in
somatic cells is one less
than number of X
chromosomes
BARR BODY
• Darkly staining
heterochromatic
mass
• Inner NM of female

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PKDIMS
squamous interphase
cells
BARR BODY
• Drum-stick mass
attached to nucleus
of neutrophils

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PKDIMS
Dr Ramakrishnan, Anatomy @
PKDIMS
BARR BODY REPORT
• [Link] female (46-XX) – positive
• [Link] male ( 46-XX) – negative
• [Link]’s syndrome (45-X0) – negative

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• [Link]’s syndrome (47-XXY) – positive
• [Link] (47-XXX) – 2 Barr body.
“Barr body – not a confirmatory test but
confirmed by Chromosomal analysis”