Advanced Techniques In Molecular Cytogenetics Karyotyping, FISH, CGH, SKY

1. 1
Presented by
Dr. Akshay S. Joshi
Advanced Techniques
In Molecular
Cytogenetics
Karyotyping, FISH, CGH, SKY

2. Introduction
History
Importance of molecular cytogenetical
techniques
Molecular cytogenetical techniques
and their applications
Conclusion

3. What Is Cytogenetics……?
Genetics - The branch of science which deals with
the study of heredity and the variation is called
as genetics.
Cytology - The branch of science which deals with
the study ofstructure and function of cell and
cell organelles is celled as cytology.
Cytogenetic - The branch of genetics which deals
with the study of cell and structure and function
of the chromosomes is called as cytogenetic.

4. Human have
tendency to
reveal the
secrets behind
every thing…..

5. Molecular
Biology
Molecular Cytogenetics
Molecular Cytogenetics
Cytogenetics
Cytology
Genetics
Molecular Cytogenetics
It involves the combination
of molecular biology and
cytogenetic. In general this
involves the use of a series
of techniques in which
DNA probes are labeled with
different colored fluorescent
tags to visualize one or more
specific regions of the
genome.

6. • The first illustration of human chromosomes
• First used the term mitosis1882
• Waldeyer introduced the term Chromosome
• Greek words for colored body
1888
• Sutton combined two disciplines, cytology
and genetics as Cytogenetics1902
• Levitsky termed The ordered arrangement
of chromosomes as karyotype1924
• Caspersson and co-workers reported a unique
banding pattern
• Drets and Shaw produces banding pattern by
Giemsa stain
1971
• Pardue and Gall demonstrated in situ
hybridization.
1969
• Tjio and Levan start human genome project1956

7. • Langer develop for the non isotope labelling
of DNA by conjugation of biotin1981
• Albertson detected biotin labelled probes1983
• Guan use chromosome microdissection for whole
chromosome painting probes (WCPs)1994
• Speicher develops multiplex fluorescence in situ
hybridization (M-FISH)
• Schröck develops a spectral karyotyping (SKY)
1996
• Tanke combined Binary RAtio labeling-FISH (COBRA FISH)
• Henegariu color-changing karyotyping (CCK)
• Chudoba developed multicolor banding (MCB)
1999
• Aurich-costa IPM-FISH2001

8. Why Molecular Cytogenetical
Techniques
• Molecular cytogenetical techniques are use to
detect chromosomal aberrations.
• There are many chromosomal aberrations are
observe in the population.

9. Causes Of Chromosomal
Abnormalities
Chromosomes Number
Chromosomes Size
The Arrangement Of
The Chromosome
Segment
No. Of Chromosome
Segment
Variation in
chromosome
morphology
Variation In

10. Variation In Chromosome Number
• A general term for the variation in the no.
• Aneuploidy
• Chromosomes no. that are not exact multiples
of n.
• It is further classified as
1. Monosomic:
2. Trisomic:
3. Tetrasomic:
4. Double trisomic:
5. Nullosomic:

11. • Euploidy / Polyploidy
• Increase in chromosomes numbers due to 1 or more
complete sets of Chromosomes is called as euploidy.
1. Monoploid:
2. Diploid:
3. Triploid:
4. Tetraploid:
5. Autotetraploid:
6. Polyploid:

12. Variation In No. Of Chromosome
Segment
• Deletion
• It is a common phenomenon of a chromosome to break.
Two types of deletions are possible
1. Terminal
2. Intercalary
• Duplication:
• Large or small piece of chromosome containing extra
blocks of genes due to duplication are found in many
individuals or even in races.

13. Variation in chromosome
morphology
1. Isochromosomes
2. Bridge-breakage –fusion-bridge cycles:
3. Ring chromosome:

14. ABNORMALITIES OF GENES/DNA
1. Point mutation
2. Gross mutation
3. Nonsense mutation
4. Silent mutation
5. Missense mutation
6. Neutral mutation
7. Frameshift mutation
8. Structural mutation

15. Genetically abnormal animals
Partial Hypotrichosis Complete Hypotrichosis
congenital alopecia and anaemia presance of marked wrinkling of the skin

16. Effect of Genetic Diseases on Livestock : an
Indian Scenario
AI
Chromosomally
abnormal bull
Use of chromosomally
abnormal semen
Infertility, repeat
breeding, genetic
disorder
Huge disaster
Dead and live sperm,
sperm motility, semen
volume
Solution
Genetic testing
of animal

17. MATING WITHOUT TESTING
NS
WITH
CARRIER
BULL
AFTER
GENETIC
TESTING
AFETR GENETIC TESTINGAFETR GENETIC TESTING
GENETICALLY CLEAN POPULATION
Consequences

18. Worldwide reports about the economic losses
due to molecular genetic disorders
• BLAD (Bovine Leukocyte Adhesion Deficiency). It was estimated that 16,000
calves were born with BLAD each year. Hence, the average economic loss per
calf was roughly 300 US dollors.
• Prevalence of BLAD in Indian HF crossbred cattle population is an alarming
situation due to consistent reports from 1999 to 2013.
• Factor XI deficiency (FXID) is responsible for repeat breeding in carriers leading
to the financial losses . In Turkey (Akyuz et al., 2012) reported an extra $ 324
financial loss compared with a normally fertile cow due to this syndrome.
• CVM ( Complex Vertebral Malformation ) carriers in India and worldwide
much more higher than other four disorders i.e. BLAD, DUMPS, Citrullinemia
and Factor XI
• In 2012 in New Zealand Livestock Improvement Corporation (LIC) , a farmer
cooperative that sells bull semen was been warned for supplying defective
semen to farmers who produced 1500 defective calves with genetic defect
leading to excessive hairiness and intolerance to heat ultimately reducing milk
producing ability.

19. Genetic disorders reported in India
• 1;29 CFT in Jersey cross cow in Chennai (Thiagarajan et al., 1990)
• 1;29 CFT in Jersey cross bull in MS used through AI for 9 years (Chauhan
et al.2009)
• 7;16 CFT in HF cross bull under use through AI in KDC Mumbai ( Biotech
lab report 1999-2000)
• 7;16 CFT in HF cross bull before use through AI in MS (Patel,1999)
• 1;9 CFTs in Gir bull in Gujarat State ( Paderi et al., 2011b)
• 16;20 CFT in Deoni bull calf in 16;20 in Karanataka State ( Paderi et al.,
2011a)
• 8;29 CFT in one Gaolao Bull and it’s 12 progeny in MS ( Umrikar et al.,
2013)

20. PREVENTION IS BETTER THAN
CURE….
!!

21. Prevention and Control
• Inherited genetic disorders affect all kinds of farm animals.
• The physiological defects arising from inherited disorders have negative
impact on health and productivity of farm animals and lead to economic
loss in the dairy cattle industry.
• Spread of undesirable genetic disorder is hastened by the artificial
insemination programme.
• Therefore, diagnosis or detection of chromosomal abnormalities is required
to diminish the risk of dissemination of recessive disorders using advance
molecular cytogenetical techniques in the dairy industry.
 The mandatory provisions of advance molecualr cytogenetical
techniques.
 The semen of every bull in commercial farms should be screened out
using molecular techniques.
 A Genetic Investigation Laboratory has been established in
Maharashtra Animal and Fishery Sciences University at Bombay
Veterinary College, Mumbai with support from Government of India
under Rashtriya Krishi Vikas Yojana in 2012.

22. Which Are The Molecular Cytogenetic
Techniques…..?
Karyotyping
Banding
Techniques
Fluroscent In Situ
Hybridization
Comparative Genomic
Hybridization
Spectral Karyotyping
Vartual Karyotyping

23. What Is Karyotyping
• The study of whole sets of chromosomes is sometimes
known as karyology.
• The chromosomes are depicted (by rearranging a
photomicrograph) in a standard format known as
a karyogram or idiogram
• Karyotyping is a test to examine chromosomes in a
sample of cells. This test can help identify genetic
problems as the cause of a disorder or disease

24. What karyotyping does…..?
• These techniques produce a characteristic pattern of
contrasting dark and light transverse bands on the
chromosomes.
• Banding makes it possible to identify homologous
chromosomes by visualization of chromosomes.
• Banding of homologous chromosomes allows chromosome
segments and rearrangements to be identified.
• it helps to detect the chromosomal abnormalities and
chromosomal alterations.
• The most widely used banding methods are G-
banding (Giemsa-banding) and R-banding (reverse-
banding).

25. How To Do Karyotyping……?
Arrange them in order.
Matched by banding patterns
Cut out
Enlarged
Picture taken
Chromosomes stained
Cell fixed in metaphase

26. Which are chromosomal banding
techniques……?
G – banding
R - banding
Q – banding
T – banding
Silver staining

27. G-banding
• G banding is obtained
with Giemsa stain following
digestion of chromosomes
with trypsin.
• It yields a series of lightly and
darkly stained bands — The dark
regions tend to be heterochromatic,
late-replicating and AT rich.
• The light regions tend to be
euchromatic, early-replicating and
GC rich.
• This method will normally produce
300–400 bands on chromosome.

28. R-banding
• is the reverse of G-banding
(the R stands for "reverse").
• The dark regions are
euchromatic (guanine-
cytosine rich regions)
• and the bright regions are
heterochromatic (thymine-
adenine rich regions).

29. Arrangement of chromosomes
• Picture shows abrupt
arrangement haploid
chromosome.
• Then scientist with
experience cut out each
chromosome and arrange
them in order according to
their size and pair.

30. Applications Of Karyotyping
• Karyotype show the chromosomal makeup of an
individual.
• Knowing the number of chromosomes is essential for
identifying chromosomal variations that cause genetic
disorders.
• Correct number of chromosomes and
chromosomal abnormality in numbers can be
detect.
• Correct size and shape of chromosomes is visualize
in this technique.
• We can identify Gender of unknown sample by
this technique.

31. Limitations Of Karyotyping
• Expert technician is require to
arrange the chromosome.
• Structural abnormalities can not be
detected by this technique.
• By observing karyograph defects like
deletion, duplication and many other
can not be detected.

32. Fluroscent in situ hybridization
(FISH)

33. What is FISH…..?
• Fluorescence in situ hybridization (FISH)
is a cytogenetic technique that
uses fluorescent probes that bind to only
those parts of the chromosome with a
high degree of sequence
complementarity
• It is used to detect and localize the
presence or absence of
specific DNA sequences on chromosomes
.
• FISH is often used for finding specific
features in DNA for use in genetic
counseling, medicine, and species
identification

34. What are probes
• probe is a synthesized fragment of DNA or RNA of
variable length which can be radioactively labeled
• The size may be varies from 100-1000 bases long.
• The probe thereby hybridizes to single-stranded nucleic
acid (DNA or RNA) whose base sequence allows probe-
target base pairing due to complementarity between the
probe and target.
• the probe is tagged (or "labeled") with a molecular
marker of either radioactive or fluorescent molecules.

35. Fluroscent In Situ Hybridization
• Step I – Denaturation
Conversion of double stranded dna in to single stranded
dna
• Step II – Hybridization
Application of probe DNA to slide & overnight incubation
at 37°C
Binding of probe DNA to trarget DNA.
• Step III – Post hybridisation washing & detection
Washing of unbound probe DNA.
Application of counter stain &
• Step IV – counter stain
Application of counter stain.
• Step V – Visualization
visualization using fluorescence microscopy.

37. Slide With Metaphase
Chromosomes
Denaturation Of DNA
Addition Of Probe
Hybridization
Washing And
Addition Of Counter
Stain
Visualization

39. Types of FISH….?
Interphase FISH
Telomeric FISH
RNA in situ
hybridization
Prime in situ
labelling
COBRA FISH
SKY FISH
M- FISH
Fibre FISH

40. Application
• FISH generally used compliment classical staining technique
• Substitute for chromosome identification at metaphase or
interphase
• Useful in several clinical settings to determine prognosis and
detection of genetic abnormalities like anupolidy,
characteristic gene fusion, cross of chromosomal region or
whole chromosom.
• Discrete information is obtained from each cell

41. • Helps in detection of single gene disorder and
presence or absence of particular gene on
chromosome.
• By using probe chromosomal material of unknown or
uncertain organism can be identified
• FISH is powerful technique use in detection of
chromosomal abnormalities
• Most significance advance in both research like gene
mapping or identification of noval oncogenes or
genetic abberation and diagnosis of haematological
malignancies and solid tumor.

42. Comparative Genomic Hybridization
(CGH)
• Comparative genomic hybridization is a molecular
cytogenetic method for analyzing copy number
variations with the help of hybridization
technique.

43. Software analysis
Fluroscence detector image analysis
Hybridization
Selection and application of probe
Isolation of DNA from Test Tissue and Reference Tissue
Metaphase Slide Preparation
Same as
FISH
CGH

44. Standard Patient Abnormal
(Probe)
Fluorescent Detector
(Probe) (Probe)

45. Duplication Deletion
Standard Abnormal Standard Abnormalnormal normal

46. Computer analysis
Software analysis
Graphical image by softwareFluorescence detector set
Fluorescence detector

47. Application of CGH
• This technology was first developed as a research tool for the investigation of
genomic alterations in cancer.
• It allows for a high-resolution evaluation of DNA copy number alterations
associated with chromosome abnormalities.
• It provides clinicians with a powerful tool to use in their increasingly
sophisticated diagnostic capabilities.
• The use of CGH is considered EXPERIMENTAL AND/OR INVESTIGATIONAL
when performed in the absence of symptoms or high risk factors for a
genetic disease or when knowledge of genetic status will not affect treatment
decisions or screening for the disease.
• Screening for prenatal gene mutations in fetuses without structural
abnormalities or testing products of conception after AI.
• Diagnosis of melanoma.
• It helps in detection of balanced rearrangements of chromosomes and for
comparison of normal and suspected DNA samples.

48. Spectral Karyotyping (SKY)

49. What is SKY…?
• Spectral karyotyping is cytogenetical technique used
to simultaneously visualize all the pairs of
chromosome in an organism in different colors.
• The sky technique is useful for identifying
chromosomal abnormalities.
• We can arrange the chromosomes according to their
number just by visualization of different colours
aquire by the chromosomes.

50. How to do SKY….?
Visualization
Image Acquisition
Washes And Detection
Application Of Dye
Hybridization
Probe Denaturation
Denaturation Of DNA
Chromosome Preparation From Tissue
Same as
FISH

51. Spectral Karyotype Observation

52. Some Abnormalities Seen In SKY
Translocation

53. Application of SKY
• This technology allows the isolation of structural
chromosome abnormalities
• which then allows determination of the precise
molecular address of chromosome breakpoints
associated with deletions translocations and insertion.
• SKY can discern the aberrations that can’t be detected
very well by conventional banding technique and
Fluorescent in situ hybridization (FISH).
• It allows visualization of all chromosomes in different
colours on same platform which is very easy to detect
chromosomal abnormalities.

54. Conclusion
• Chromosomal genetic abnormalities are the hidden
cause for the huge economic losses.
• Molecular cytogenetic techniques like FISH, CGH
and SKY are the available advanced diagnostic
tools to detect such chromosomal abnormalities
and to prevent the spread in the population which
indirectly avoid the economic consequences.

55. References
Ashutosh H., M. Jain, I. Chaudhary, N. Gupta and M. Kabra 2013 Fluorescence in situ hybridization (FISH) using non-commercial probes in
the diagnosis of clinically suspected microdeletion syndromes. Indian J Med Res 138, pp 135-142.
Christine M. (2008) Spectral Karyotyping (Sky) Establishes Chromosomal Homologies Between A New World Monkey (Pithecia Pithecia)
And Humans (Homo Sapiens). TCNJ Journal Volume X : 1 – 12.
Emanuela V. V. and J. M. Bridger (2008) FISH glossary: an overview of the fluorescence in situ hybridization technique. BioTechniques Vol.
45 (4): 385 – 405.
George I. and O. Arisaka (2012) Chromosome Analysis Using Spectral Karyotyping (SKY). Cell Biochem Biophys 62:13–17.
Jeffrey M. and R. H. Singer (2003) Fluorescence in situ hybridization: past, present and future. Journal of Cell Science 116 (14) 2833 – 2838.
Kakazu N. and T. Abe (2006) Multicolor banding technique, spectral color banding (SCAN): new development and applications. Cytogenet
Genome Res 114: 250–256.
Malcolm A. F. S. (2015) History and evolution of cytogenetics. Molecular Cytogenetics 8 (19): 2– 8.
R. Raff and G. Schwanitz (2001) Fluorescence in situ Hybridization General Principles and Clinical Application with Special Emphasis to
Interphase Diagnostics. IJHG 1(1): 65-75.
Regine S., E. Schrock, T. Ried and G. Schwanitz (2004) Spectral Karyotyping (SKY) permits the Characterisation of a de Novo unbalanced
Translocation 46, XY, der(14)t(12;14). Int J Hum Genet, 4 (3): 173-178.
Wendy A. B. 2001 Karyotype Analysis and Chromosome Banding Encyclopedia Of Life Sciences/ & 2001 Nature Publishing Group /
www.els.net.
Yuval G., I. T. Young and G. McNamara (2006) Spectral Imaging: Principles and Applications. Cytometry Part A 69: 735–747.