A cytological technique to detect the nature of adjacent chromosomal regions by using different staining technique assisted with some pre treatment of metaphase chromosomes prepared on the slides

1. Chromosomal Banding
Technique
Kishore Kumar Gupta
Associate Professor
University Department of zoology

2. • A cytological techniques to reveal the nature of adjacent
regions of the chromosomes through out their length in
form of dark and light bands .
• Karyotpe preparation is the essential initial steps
• Karyotype refers to chromosomal; constituents of cells of
an organisms
• Use to detect the number , size and shape of the
chromosomes
• 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”.

3. • Tissue culture is a long term or short term technique to
understand the various molecular , cellular and organic
functions
• Short time Lymphocyte culture is the simplest form by
using lectin as mitigen like phytohaemagglutinin
(PHA),concanavilin A pokeweed mitogen etc.
• Use to detect chromosomal abnormality and
chromosomal rearrangements
• Somatic cell fusion and hybridization for gene mapping

4. Protocol
• Add 0.3ml blood+5ml TC medium +1ml Foetal bovine
serum+ 0.1ml PHA
• Buffer the pH , blow CO2n from CO2 cylinder or bubbled
exhaled air orally through a cotton plugged pipette
• Kept in incubator at 37 deg celcius for 4b hrs to 72 hrs
• Inspect culture every morning and evening for the change
in pH and infection and shaken to break the clumps of RBC
• Prior to chromosomal preparation cocemid
(0.02microgram/ml was added
RMPI + L Glutamine

5. Karyotyping
• Centrifuge at 1000-1200rpm for 5 min.
• Decant supernatant and fresh suspension in pre-warmed 0.56 %kCl(hypotonic)
with initial small volume and then make 8-10ml ,
• Keep in incubator for 18-20 min at 37 deg celcius
• Add 3-4 drops of fixative (1:3 – Acetic acid : Methanol and spin (1000-1200 RpM .
• Decant hypotonic completely
• Add fixative drop by drop(3-4) to fix the cells and then suspension was made to
the volume to 8-10 ml . Keep for 15 min
• Recentrifuge at 1000-2000 RPM for 5 moin , decant , resupend pellets in 8-10 ml
fixative . Keep for 10 min.
• recentrifuge and resuspend in -.2 -0.4 ml fixative.
• Agitate suspends
• Wipe a slide with 70% alcohasl .wipe ant prewarmed the slide on hot plate
• One Drop suspension and expose to flame for air drying or let it be in air
• Stain with giemsa ( Giemsa Pwder 380 mg + 25 ml metanol+ gycerol for stock)
• Working Stock 2.5 ml+ metanol1.5 ml + 50 ml Giemsa water ( 0.2 M Na2 HPO4 80
ml+ 800 ml DW)

6. Protocol

7. Types of Karyotype
• Asymmetric Karyotype- Show larger difference
between smaller and larger chromosome in a
set., Have more acrocentric chromosomes.
Symmetric Karyotype - Show lesser difference
between smaller and larger chromosome in a
set. Have more metacentric chromosomes

8. •Chromosmes has domain which
manifest various structural and
functional attributes of chromatin .
•Distinct chromatins are Euchromatin
and heterochromatin ( Facultative and
constitutive)
•At interphase EC is decondensed and
active genomic part with unique
sequences and HC is condensed (
transcriptional inert) and consists
repeated sequences
•both domain tends to vary from species
to species and co-relate with the nature
of gene i.e distribution of the nucleotide
sequences.
•Chromatin nature can be visualized as
band after staining with different dyes
and is of significance in many studies

9. Why Banding ?
It allows to judge changes in smaller pieces of the chromosomes
and related abnormalities which are not visible in analysis
Representation of cytogenetic bands
The cytogenetic bands are labled as P1 P2 P3 ,q1 q2 etc from the centromere to
telomere . At high resolution , sub band and sub sub bands are seen and
accordingly numbered as
Example 1
Locus 7q31.2 – indicates gene on 7th log arms at band 3, sub band 1 and sub
sub band 2
7qtel – 7th , long at telomere

10. Thus …
• Chromosome banding techniques produce a
series of consistent landmarks along the length of
metaphase chromosomes that allow for both
recognition of individual chromosomes within a
genome and identification of specific segments of
individual chromosomes.
• These landmarks facilitate assessment of
chromosome normalcy, identification of sites of
chromosome breaks and alterations, and location
of specific genes.

11. Types & applications

12. Important Bandings
Banding
Q banding
G banding
R Banding
C banding
H banding
R Banding

13. Q banding
• QUINACRINE BANDING (Q-BANDING)
• Q-banding (QFQ) was the first chromosome-banding method (Caspersson
et al., 1970)and remains the simplest.
• Slides of metaphase chromosomes are exposed to the fluorescent
• DNA intercalating agent quinacrine and viewed with a fluorescence
microscope
• Quinacrine, when bound to DNA, absorbs light at∼455 nm and has a
maximal emission at ∼495 nm. Hence, excitation filters in the 430- to 460-
nm range.
• Materials
• Air-dried slides of metaphase chromosomes
• Quinacrine staining solution
• McIlvaine buffer, pH 5.6
• Immersion oil, low fluorescence
• Coverslips, no. 0 or no. 1
McIlvaine buffer, pH 7.0
18.1 ml 0.1 M citric acid
81.9 ml 0.2 M dibasic sodium
phosphate (Na2HPO4)
Check pH and adjust to 7.0 with
either solution, if necessary
Store ≤6 months at room
temperature

14. Protocol for Q banding
• 1.Place air-dried slide of metaphase chromosomes in a
Couplin jar containing quinacrine staining solution, 5
min at room temperature.
• 2. Rinse slide by dipping several times into Coplin jar
filled with water. Discard rinse water, refill jar with with
fresh water, and repeat rinse. Air dry slide.
• Slide can be stored dry after this step for weeksin
backellite box to protect from light.
• 3. Mount slide using McIlvaine buffer, pH 5.6.
• Add a coverslip and gently squeeze excess buffer from
under coverslip by blotting gently with paper towel.

15. GTG Technique for G-Banding
• Materials
• Trypsin solution (20mg/ml in 0.9% NaCl
sored at -20 deg celcius)
• 70% and 90% (v/v) ethanol
• 7-10% Giemsa staining solution
Protocol
• Thaw 1ml trypsin and make 50 ml with 0.9%
NaCl in a couplin Jar ( pH 7.,5- 7.8)
• Add 50 ml phosphate buffer
• In another couplin jar, prepare 7-10%
Giemsa stain
• Dip slides in trypsin for 5 sec.
• Rinse in phophate buffer
• stain in giemsa for 3-5 min.

16. R Banding: Reverse chromosome banding
• (R-banding) produces a pattern of bands that is the reverse of the Q- and
G-banding pattern—i.e., a dark (positive) G-band is a light (negative) R-
band and vice versa.
• R-banding can be used for chromosome identification, although G-banding
is usually preferred.
• R-banding is also useful for visualization of telomeric ends of
chromosomes; these ends stain intensely with R-banding and negatively
with G-banding
RHG Technique for R-Banding
1. Preheat 25 M sodium phosphate buffer (pH 4.0) to 87° or 88°C in covered Coplin
jars in covered circulating water bath.
Both Coplin jars and water bath should be kept covered to maintain temperature.
2. Prewet each air-dried slide of metaphase chromosomes in water and place in
87° to 88°C sodium phosphate buffer (pH 4.0), one slide per jar, 5 to 15 min. Rinse
in water.
3. Stain slide 5 to 10 min in 10% Giemsa staining solution. Rinse in water and air
dry.

17. CA3/DA Technique for R-Banding
• 1. Apply 100 μl chromomycin A3 staining solution to air-dried metaphase
chromosome slide and cover with no. 1 coverslip. Allow to stain 15 min.
• 2. Remove coverslip, then rinse by quickly pouring water over slide. Shake off
excess water.
• 3. Apply 100 μl distamycin A staining solution to slide and cover with no. 1
coverslip. Allow to stain 5 min.
• 4. Remove coverslip and rinse in distilled water as in step 2.
• 5. Mount in glycerol and add a coverslip.
• 6. Examine and photograph with fluorescence microscope with appropriate
• filter set
Distamycin A staining solution with
MgCl2
1 mg distamycin A (Sigma)
5 ml McIlvaine buffer, pH 7.0
5 ml H2O
1 mg MgCl2⋅6H2O
Store ≤1 month at −20°C in 1.5-ml aliquots
wrapped in foil
Chromomycin A3 staining solution
Dissolve 5 mg chromomycin in two
drops 100% ethanol. Add 10 ml of a
solution containing 5 ml McIlvaine
buffer, pH 7.0, 5 ml H2O, and 1mg (5
mM) of MgCl2⋅6H2O.
Mix. Store in 1.5-ml aliquots ≤1 year at
−20°C wrapped in foil.
Discard thawed solution after 2 weeks.

19. 5%Ba(OH)2
Dip the slides in 0.2NHCl in a couplin jar for 30 ‘ followed by
two rinse in DW
Air dry and put in Ba ( OH)2 at 50 deg celcius for 1 to 5 ‘
Rinse in two for change in DW
Dry and keep in 2XSSC inside water bath at 60 deg celcius fotr
1-2hrs
Rinse in water and stain with giemsa for 15-20 mi. Mount in
DPX

20. N Banding – Detection of NOR region
This procedure is sometimes referred to as silver staining because it uses a
silver nitrate solution.
It stains the active ribosomal DNA-containing nucleolar organizer regions
in interphase nuclei and sites near the ends of the short arms (in the area
called the stalks) of human acrocentric chromosomes 13, 14, 15, 21, and
22. This stain is viewed with a
bright-field microscope
PROTOCOL
1. Add 3 drops of 2% gelatin solution and 4 drops of 50% silver nitrate
solution to recently made, unheated, air-dried slide of metaphase
chromosomes. Cover with
coverslip.
2. Heat 2 to 4 min on 65°C slide warmer or in drying oven. Remove slide
from heat when solution appears golden brown.
3. Rinse in beaker with ∼100 ml of 3% acetic acid.
Acetic acid works like a stop bath to end the staining reaction.
4. Rinse in water and air dry.
5. View and photograph with bright-field microscope

21. NOR on chromosomes 13,14
15,21 & 22
N banding