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Presented byDr. Akshay S. Joshi

Advanced Techniques In

Molecular CytogeneticsKaryotyping, FISH, CGH, SKY

IndexIntroduction

History

Importance of molecular cytogenetical techniques

Molecular cytogenetical techniques and their applications

Conclusion

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.

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

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.

1882 •The first illustration of human chromosomes •First used the term mitosis

1888• Waldeyer introduced the term

Chromosome• Greek words for colored body

1902 • Sutton combined two disciplines, cytology and genetics as Cytogenetics

1924 • Levitsky termed The ordered arrangement of chromosomes as karyotype

1971• Caspersson and co-workers reported a

unique banding pattern• Drets and Shaw produces banding pattern

by Giemsa stain

1969 • Pardue and Gall demonstrated in situ hybridization.

• Tjio and Levan start human genome project1956

1981 •Langer develop for the non isotope labelling of DNA by conjugation of biotin

1983 •Albertson detected biotin labelled probes

1994• Guan use chromosome microdissection for

whole chromosome painting probes (WCPs)

1996 • Speicher develops multiplex fluorescence in situ hybridization (M-FISH)

• Schröck develops a spectral karyotyping (SKY)

• Tanke combined Binary RAtio labeling-FISH (COBRA FISH) • Henegariu color-changing karyotyping (CCK)• Chudoba developed multicolor banding (MCB)

1999

• Aurich-costa IPM-FISH2001

Why Molecular Cytogenetical Techniques

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

Causes Of Chromosomal Abnormalities

Chromosomes Number

Chromosomes Size

The Arrangement Of The

Chromosome Segment No. Of

Chromosome Segment

Variation in chromosome morphology

Variation In

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 as1. Monosomic: 2. Trisomic:3. Tetrasomic:4. Double trisomic: 5. Nullosomic:

• 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:

Variation In No. Of Chromosome Segment

• Deletion• It is a common phenomenon of a chromosome

to break. Two types of deletions are possible1. Terminal2. 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.

Variation in chromosome morphology

1. Isochromosomes2. Bridge-breakage –fusion-bridge

cycles: 3. Ring chromosome:

ABNORMALITIES OF GENES/DNA1. Point mutation2. Gross mutation3. Nonsense mutation4. Silent mutation5. Missense mutation6. Neutral mutation7. Frameshift

mutation8. Structural mutation

Genetically abnormal animals

Partial Hypotrichosis Complete Hypotrichosis

congenital alopecia and anaemia presance of marked wrinkling of the skin

Effect of Genetic Diseases on Livestock : an Indian Scenario

AIChromosomally 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

MATING WITHOUT TESTING

NS WITH CARRIER     BULL

AFTER GENETIC TESTING                 

AFETR GENETIC TESTINGAFETR GENETIC TESTING

GENETICALLY CLEAN POPULATION

Consequences

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.

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)

PREVENTION IS BETTER THAN CURE….!!

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.

Which Are The Molecular Cytogenetic Techniques…..?

KaryotypingBanding

TechniquesFluroscent In Situ

HybridizationComparative

Genomic HybridizationSpectral

KaryotypingVartual Karyotyping

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

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).

How To Do Karyotyping……?

Arrange them in order.

Matched by banding patterns

Cut out

Enlarged

Picture taken

Chromosomes stained

Cell fixed in metaphase

Which are chromosomal banding techniques……?

G – banding

R - banding

Q – banding

T – banding

Silver staining

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.

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).

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.

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.

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. I AM

EXPERT

Fluroscent in situ hybridization(FISH)

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

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.

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°CBinding of probe DNA to trarget DNA.

• Step III – Post hybridisation washing & detection

Washing of unbound probe DNA.Application of counter stain &

• Step IV – counter stainApplication of counter stain.

• Step V – Visualizationvisualization using fluorescence microscopy.

Slide With Metaphase Chromosomes

Denaturation Of DNA

Addition Of Probe

Hybridization

Washing And Addition Of Counter

Stain

Visualization

Types of FISH….?Interphase

FISH

Telomeric FISHRNA in situ

hybridizationPrime in situ

labellingCOBRA FISH

SKY FISH

M- FISHFibre FISH

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

• 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.

Comparative Genomic Hybridization (CGH)

• Comparative genomic hybridization is a molecular cytogenetic method for analyzing copy number variations with the help of hybridization technique.

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

Standard Patient Abnormal

(Probe)

Fluorescent Detector

(Probe) (Probe)

Duplication

DeletionStandard Abnormal Standard Abnormalnorma

lnormal

Computer analysis

Software analysis

Graphical image by softwareFluorescence detector set

Fluorescence detector

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.

Spectral Karyotyping (SKY)

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.

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

Spectral Karyotype Observation

Some Abnormalities Seen In SKY

Translocation

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.

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.

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.