1. Genetic Series:
Cytogenetics
Dr Mathew Joseph
MBBS,MD(AIIMS),BCC(Palliative Medicine)
Assistant Professor
Department of Anatomy
Amala Institute of Medical Sciences, Thrissur

2. • Cytogenetics is a branch of genetics that studies the number and
structure of the chromosomes.
• Cytogenetic study involves following methods:
– Karyotyping
– Fluorescent in situ hybridization (FISH)
– Buccal smear for fluorescent bodies
– Study of sex chromatin or Barr bodies
– Study of drumstick body in neutrophils.
INTRODUCTION

3. Q. Write a short note on karyotyping.
• Karyotype – Complete chromosome set of a cell.
• Karyotyping – Method of obtaining a karyotype of a cell.
• For chromosome analysis, the karyotype is prepared from the
arrested cell division at metaphase
KARYOTYPING

4. Steps for karyotyping

5. 1. Sample collection: Blood, chorionic villus sample, amniotic fluid, bone marrow or
fetal blood is collected in heparinized vacutainer.
2. Culture setup: Cells from collected sample are transferred to culture media (RPMI
1640 or TC 199) containing fetal bovine serum (nutrition),
phytohemagglutinin (mitotic agent) and antibiotics (to prevent
bacterial growth).
3. Incubation: Cultured cells are incubated at 37°C for 72 hours to allow cells to
undergo mitosis.
4. Colchicine block: Small amount of colchicine is added to the culture to block cell
division in metaphase.
4. Harvesting: Cultured cells are washed with a hypotonic solution (to remove red
blood cells and to swell all the cells). These cells are fixed with chilled
fixative (a mixture of glacial acetic acid and methanol).
PROTOCOL FOR KARYOTYPING

6. 5. Slide preparation: Slide is prepared from the drop of fixed cells.
6. Staining: Slide is stained with Giemsa stain, G-banding (, Q-
banding, R-banding, C-banding or nucleolus organizer
region-staining.
7. Karyotyping: Stained slide is observed under a microscope and
microphotographed, and chromosomes are arranged.
PROTOCOL FOR KARYOTYPING

7. • Photographed chromosomes are arranged in the following manner:
– Length of chromosome
– Position of centromere
– Presence of satellite body
– Presence of specific bands.
• According to the Denver classification, chromosomes are arranged
into seven groups A–G
• While arranging karyotype, all chromosomes are arranged in pairs in
their corresponding group, except X and Y that are arranged at the end
(X is followed by Y).
KARYOTYPING

8. Normal male karyotype (46,XY).

9. Normal female karyotype (46,XX)

10. Group Chromosome pair Features
A 1, 2, 3 1 and 3– metacentric,
2–submetacentric
B 4, 5 Submetacentric
C 6–12, X Submetacentric
D 13–15 Acrocentric
E 16–18 16–metacentric,
17, 18–submetacentric
F 19–20 Metacentric
G 21–22, Y Acrocentric
Denver classification of chromosomes.

11. It is also known Giemsa banding.
Most common employed procedure for karyotyping
Need of banding method:
• To differentiate between chromosomes of the same group, for example,
chromosomes 4 and 5.
• To identify addition, deletion, inversion, and translocations.
Procedure:
• Slide of chromosomes are treated for 10 to 20 seconds with trypsin (to
partially digest histone proteins) before Giemsa staining
↓
• Densely stained region of chromosome–dark band (A-T rich)
• Lightly stained region of chromosome–light band (G-C rich).
G-banding

12. • Clinical diagnosis of cases with chromosomal aberrations.
• Clinical confirmation of cancer cases showing translocations, for
example, detection of Philadelphia chromosome t(9;22)(q34;q11.2) in
chronic myeloid leukemia.
• Prenatal diagnosis for chromosomal aberrations.
• Diagnosis of chromosomal defects in infertility cases or in recurrent
abortion cases.
• Note: For detection of single nucleotide change in DNA, polymerase
chain reaction is used rather than cytogenetics.
Uses of Karyotyping

13. .
• Band is a part of chromosome which is clearly distinguishable from its
adjacent segment by lighter or darker with various banding methods.
• Q-banding: Chromosomes are stained with fluorescent dye—quinacrine
and it is useful for studying heterochromatin of chromosomes 1, 9 and 16.
•
• R-banding: Slides are preheated before Giemsa staining to generate the
reverse G-banding pattern (G-C rich dark bands).
• C-banding: DNA of the chromosome is denatured with acid, alkali or heat
prior to Giemsa staining. It is useful for studying constitutive
heterochromatin (dark banding).
Some Interesting Facts

14. • Fluorescent in situ hybridization (FISH) is a cytogenic technique that utilises
the property of DNA probe to bind with its complementary target DNA
sequence.
• DNA probes are tagged with fluorescent substances (may be with radioactive
labels)
Procedure
: Cells collected from tissue → culture → colchicine block →
harvesting → metaphase preparation → hybridization with
DNA probe → observation under a fluorescent microscope.
Cells collected from tissue → slide preparation → hybridization with
DNA probe → observation under a fluorescent microscope.
FLUORESCENT IN SITU HYBRIDIZATION

15. :
• To confirm the presence of specific chromosome or gene.
• To assess structural defects of chromosomes such as translocation.
• To identify small deletion or addition of chromosome.
FLUORESCENT IN SITU HYBRIDIZATION

16. Feulgen reaction:
It is a staining method for cellular DNA that reacts with Schiff’s reagent. It is specific for 2-deoxyribose
sugar.
Spectral karyotyping (SKY):
•It is a cytogenetic technique that involves simultaneous painting of all human chromosome with
different fluorescent-colored DNA probe.
•SKY technique is useful for identification of multiple translocations.
High-resolution banding (HRB) technique:
•In chromosome banding technique, total number of bands in all chromosomes is called as band level.
•Chromosomes in metaphase are condensed → hence can produce only 200–350 bands.
•For detailed analysis, longer chromosomes are obtained from late prophase or early metaphase.
•Procedure: Similar as karyotyping except treatment with ethidium bromide, actinomycin D or Hoechst
with short exposure of colchicine.
•Use: To obtain 800–1400 band level for easy diagnosis of deletion, addition, translocation and many
other chromosomal aberrations.
Feulgen reaction, SKY, HRB

17. Thank You