Karyotyping and Chromosome

Banding Technique
 Introduction
• Identification of chromosome that differs
morphologically.
• Banding techniques are based on
identification of chromosome segment that
consists of rich AT & GC region.
• 1st step in this is denaturation followed by
slow renaturation. During slow renaturation
the heterochromatin region can be identified
which consists of repetitive DNA sequances.
 Karyotype
The karyotype is the complete chromosomal
set of the nucleus of the cell. A diagrammatic
representation of the karyotype with all of the
pairs of the chromosomes arranged in order of
size is called an ideogram.

Every organism has a standered karyotype,

which provides a frame of reference for the
analysis of mutations.
• Karyotype analysis is the study of all the
visible traits of chromosomes in a typical cell.
This is useful in studying speciation.

• Ex1: Distinict species of drosophila that are

very similar in appearnce are distinquished
easily by the karyotypic analysis, which reveals
numerous chromosoma; inversion and
translocation.
 Chromosome Banding
• Chromosome banding pattern is composed of
alternating light and dark stripes, or bands
that appear along its length after being
stained with a dye.

• A unique banding pattern is used to identify

each chromosome and to diagnose
chromosomal aberrations.
 Chromosome Banding Cont....
• In the 1950s, chromosomes from the cell’s
nucleus were identified with a uniform
(Unbaneded) stained that allowed for the
observation of the overall length and primary
constriction (Centromere) of each chromosome
as well as a secondary constriction in
chromosome.
• The staining techniques used to make the bands
visible were developed in the late 1960s and
early 1970s
 Banding of Chromosomes
• G - Banding
• Q - Banding
• C - Banding
• R - Banding
• T - Banding
• NOR - Banding
• High Resolution Banding
• Restriction Endonuclease Banding
 Staining
Banding patterns can be visually identified on
chromosomes after staining.

Traditional Types
• G-Banding – Giemsa stain
• Q-Banding – Fluorescent stain
• R-Banding – Reverse Giemsa stain

New Type
• Fluorescence In Situ Hybridization techniques
 TERMS AND DEFINITIONS OF VARIOUS
ABERRATIONS OF CHROMOSOMES
• Ring( r) • Hyperdiploid (h)
• Dicentric (d) • Chromatid deletion (td)
• Chromosome gap (sg) • Acentric fragment (af)
• Fragment (f) • Triradial (tr)
• Translocation (t) • Pulverized chromosome (pu)
• Quadriradial (qr)
• Pulverized chromosome
(pu+)
• Pulverized cell (puc)
• Complex rearrangement (cr)
• Polyploid (pp) or
endoreduplication
• Minute (min)
 G-Banding Technique
• Giemsa is the most commaonly used stain in
cytogenetic alalysis.
• Staining a metaphase chromosome with a
Giemsa stain is referred to as G- Banding
• Most G-banding techniques require
pretreating .
• Chromosomes are pre-treated with either salt
or a proteolytic (protein-digesting)
 G-Banding Technique cont....
• “GTG banding” refers to the process in which
G-banding is preceded by treating
chromosome with trypsin.
• G-banding preferentially stains the regions of
DNA that are rich in adenine and thymine.
• The regions of the chromosome that are rich
in guanine and cytosine have little affinity for
the dye and remain light.
 G-Banding Technique cont......
• Slandered G-band staining techniques allow
between 400 and 600 bands to be on
metaphase chromosomes.

• With height-resolution G-banding techniques,

as many as two thousand different bands have
been catalogued on the twenty-four human
chromosome
G- banding technique methodology
• Ageing of good slides for 10 days
• Normal saline
• Treated with trypsin 0.25% solution 10-15 sec
• Immersed in 70% ethanol for few minutes
• Stained with 10% Giemsa for 6-10min
• Microphotograph good spreads
• Construction of G-banded karyotype
 G-BANDED
KARYOTYPE
OF CATTLE
 C-banding Methadology
C banding:
Chromosomes are treated with acid and base, then
stained with Giesma stain
1.Treat the slides in 0.2 N HCI for one hr at room
temperature.
2. Rinse in de-ionized water.
3. Immerse in 1% barium hydroxide at 50°C for 5-15 min.
4. Rinse in de-ionized water.
5. Incubate at 60°C in 2XSSC buffer for one hr.
6. Rinse in de-ionized water and stain in 4% Giemsa stain
for 90 min.
7. Rinse in de-ionized water, dry and examine under oil
immersion.
• C-banding stains areas of heterochromatin,
which is tightly packed and repetitive DNA. C-
banding is specifically useful in humans to
stain the centromeric chromosome regions and
other regions containing constitutive
heterochromatin - secondary constrictions of
human chromosomes 1, 9, 16, and the distal
segment of the Y chromosome long arm.
 NOR-banding
• NOR-banding involves silver staining (silver
nitrate solution) of the "nucleolar organizing
region", which contains rRNA genes.

• AgNO3 (Silver Nitrate) which binds to NOR

region. The band represents non-histone
protein which specifically at NOR region