ppt on karyotyping, chromosome banding and chromosome painting

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Page 1: Ppt on karyotyping, chromosome banding and chromosome painting

WELCOME

Page 2: Ppt on karyotyping, chromosome banding and chromosome painting

Karyotyping, Chromosome Banding and Chromosome Painting

Page 3: Ppt on karyotyping, chromosome banding and chromosome painting

1.Karyotyping

Page 4: Ppt on karyotyping, chromosome banding and chromosome painting

Introduction• Particular chromosome complement of an individual or a

related group of individuals, as defined by the chromosome size, morphology and number is known as a “Karyotype”.

• KaryotypeSize of chromosomePosition of centromerePresence of secondary constrictionSize of satellite

Page 5: Ppt on karyotyping, chromosome banding and chromosome painting

• Derived from Greek word “karyon”, which means "nucleus”, karyotype is represented as Idiogram.

• When the haploid set of chromosomes of an organism are ordered in a series of decreasing size, it is said to be an idiogram.

• In other sense diagrammatic representation of a karyotype is an Idiogram.

Page 6: Ppt on karyotyping, chromosome banding and chromosome painting

History of karyotyping

• 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”.

Page 7: Ppt on karyotyping, chromosome banding and chromosome painting

Types of Karyotype

Karyotype

Symmetric Karyotype

Asymmetric Karyotype

Page 8: Ppt on karyotyping, chromosome banding and chromosome painting

Types of Karyotype

Asymmetric Karyotype• Show larger difference

between smaller and larger chromosome in a set.

• Have more acrocentric chromosomes.

• Have relatively advanced feature

Symmetric Karyotype• Show lesser difference

between smaller and larger chromosome in a set.

• Have more metacentric chromosomes.• Have no relatively

advanced feature

Page 9: Ppt on karyotyping, chromosome banding and chromosome painting

• In 1931 G.A. Levitzky, a Russian scientist suggested that in flowering plants there is a predominant trend towards karyotype asymmetry. This trend has been carefully studied in the genus Crepis of the family compositae.

Page 10: Ppt on karyotyping, chromosome banding and chromosome painting
Page 11: Ppt on karyotyping, chromosome banding and chromosome painting

Species showing a greater asymmetry is more advanced. HOW???

Degree of asymmetry• Proportion of metacentric, acrocentric

chromosomes in a set.• Ratio between size of largest and smallest

chromosomes in a set. Interpretation• Higher the proportion of acrocentric

chromosomes, Greater the value of size ratio, more asymmetrical is a karyotype

Page 12: Ppt on karyotyping, chromosome banding and chromosome painting

Procedure of karyotyping

Page 13: Ppt on karyotyping, chromosome banding and chromosome painting

Process of karyotyping in Plants

Plant

Root tips -0.5 to 1cm

Pretreated with colchicine for 3 hrs at 26 ᵒC

Slide preparation

N banding technique

Cells atMetaphase

Page 14: Ppt on karyotyping, chromosome banding and chromosome painting

Representation of a karyotype

• By arranging chromosomes of somatic complement in a descending order of size keeping their centromeres in a straight line.

Longest chromosome –on extreme left.Shortest chromosome –on extreme rightSex chromosomes –allosomes–extreme right

Page 15: Ppt on karyotyping, chromosome banding and chromosome painting
Page 16: Ppt on karyotyping, chromosome banding and chromosome painting

Advantages of Karyotyping

• Reveals structural features of each chromosomes.

• Helps in studying chromosome banding pattern.

• Helps in the identification of chromosomal aberrations.

• Diagnosis of prenatal genetic defects.• Aids in studying evolutionary changes .

Page 17: Ppt on karyotyping, chromosome banding and chromosome painting

2.Chromosome Banding

Page 18: Ppt on karyotyping, chromosome banding and chromosome painting

Introduction

Page 19: Ppt on karyotyping, chromosome banding and chromosome painting

History

Page 20: Ppt on karyotyping, chromosome banding and chromosome painting

Why to study Banding pattern??

• This allows you to see smaller pieces of the chromosome, so that you could identify smaller structural chromosome abnormalities not visible on a routine analysis.

Page 21: Ppt on karyotyping, chromosome banding and chromosome painting

Classification of Banding Techniques

Based onGC and AT rich regions.Constitutive Heterochromatin Region.

Always metaphase chromosomes whose size has condensed and whose diameter is increased are used for chromosome banding studies after fixing the stage.

Page 22: Ppt on karyotyping, chromosome banding and chromosome painting

Banding Techniques

Q(Quinarcine)

G (Giemsa) N (NOR)

C(Centromeric)

1958 1971 1973 1978

Casperson et.al

Summer et.al

Matsui & Sasaki

Linde &Laursen

Page 23: Ppt on karyotyping, chromosome banding and chromosome painting

1.Q Banding TechniquesChromosome

Stained with Quinarcine Mustard

Subjected UV light

Banding Pattern

Region rich in GC bases

Region Rich in AT bases

Light stainingDark staining

GC region quenches dye but do not fluorescence ,situated in

euchromatin region

AT region quenches dye & fluorescence, situated in heterochromatin region

Page 24: Ppt on karyotyping, chromosome banding and chromosome painting

Q Banding Techniques

Advantages

• Simple and Versatile.• Used where G band is

not accepted.• Used in study of

chromosome heteromorphism.

Disadvantages• Tendency to fade during

examination. Photo-degradation . Chromopore- absorb

light of a particular wavelength due to a chemical bond formed between dye and light.

UV light breaks the chemical bond.

Page 25: Ppt on karyotyping, chromosome banding and chromosome painting

2.G Banding TechniquesChromosome

Weak Trypsin / urea/ protease

Treated with Giemsa

Banding pattern

To denatureprotein

Interaction of the DNA with

thiazine & eosin components of stain brightens

sulphur rich regions

Methylene Azure+Methylene Violet+ Methylene Blue+ Eosine

Page 26: Ppt on karyotyping, chromosome banding and chromosome painting

G Banding Techniques

Advantages• Used in identification of

bands rich in Sulphur content.

• Used in the identification of chromosomal abnormalities

• Gene Mapping.

Disadvantages• Not used in plants.

Page 27: Ppt on karyotyping, chromosome banding and chromosome painting

G banding not used in Plants. Why????

• Human mitotic metaphase chromosome is 2.3 times shorter.

• Plant mitotic metaphase chromosome is 10 times more shorter than human chromosome.

• Hence difficult to demonstrate the arrangement of bands at this level of saturation with G banding technique.

Source: Greilhuber, J (1977). Why Plant chromosome do not show G bands? .Theory of Applied Genetics, Vol 50(3): 121- 124.

Page 28: Ppt on karyotyping, chromosome banding and chromosome painting

3. N Banding TechniquesChromosome

Air Dried

Treated with 5% Trichloroacetic acid @ 95ᵒC for 30 min.

Treated with 0.1N HCl @ 60ᵒC for 30 min.

Banding pattern in Structural non- histone proteins linked to NOR

region

Page 29: Ppt on karyotyping, chromosome banding and chromosome painting

N Banding Techniques

Advantages• Used in the identification of Nucleolar

organizer region.• Superior banding pattern for plants.

Page 30: Ppt on karyotyping, chromosome banding and chromosome painting

4. C Banding Techniques

Chromosome

Treating with alkali solution

Washing with Sodium citrate @ 60ᵒC for 30 min.

Staining with Giemsa Solution

Banding pattern at heterochromatin region

Repeatitive DNA renature but

unique DNA do not renature

DNA denaturing

Page 31: Ppt on karyotyping, chromosome banding and chromosome painting

C Banding Techniques

Advantages• Identification of chromosomes particularly in

insects and plants.• Identification of bivalents at diakinesis using

both centromere position.• Paternity testing.• Gene mapping.

Page 32: Ppt on karyotyping, chromosome banding and chromosome painting

Representation of a Chromosome Band

• Each human chromosome has a short arm ("p" for "petit") and long arm ("q" for "queue"), separated by a centromere.

• Each chromosome arm is divided into regions, or cytogenetic bands, that can be seen using a microscope and special stains.

Page 33: Ppt on karyotyping, chromosome banding and chromosome painting

• The cytogenetic bands are labeled p1, p2, q1, q2, etc., counting from the centromere out toward the telomeres.

• At higher resolutions, sub-bands can be seen within the bands. The sub-bands are also numbered from the centromere out toward the telomere.

Page 34: Ppt on karyotyping, chromosome banding and chromosome painting

• Example 1: The cytogenetic map location of a gene is 7q31.2.

This indicates that the gene is on chromosome 7, q arm, band 3, sub-band 1, and sub-sub-band 2.

• The ends of the chromosomes are labeled ptel and qtel.

• Example 2: The notation 7qtel refers to the end of the long arm of chromosome 7.

Page 35: Ppt on karyotyping, chromosome banding and chromosome painting
Page 36: Ppt on karyotyping, chromosome banding and chromosome painting
Page 37: Ppt on karyotyping, chromosome banding and chromosome painting

3.Chromosome Painting

Page 38: Ppt on karyotyping, chromosome banding and chromosome painting

Introduction

• Chromosome 'painting' refers to the hybridization of fluorescently labeled chromosome-specific, composite probe pools to cytological preparations.

• First termed by Pinkel et.al in 1988.• Chromosome painting coupled with

Fluorescence in situ hybridization (FISH) is used routinely for identification of chromosomes.

Page 39: Ppt on karyotyping, chromosome banding and chromosome painting

WHY?????

Helps in the identification of Chromosomal rearrangements.

Helps in the identification of Chromosomal breakpoints.

Helps in determination of extra chromosomal material.

Page 40: Ppt on karyotyping, chromosome banding and chromosome painting

Procedures Sample preparation and hybridization

• Prepare slides with metaphase chromosomes .• Dehydrate in ethanol.• Denature DNA at 70ᵒC.• Denature labeled probe.• Incubate at 37ᵒC for 4-16 hours for hybridization.

Page 41: Ppt on karyotyping, chromosome banding and chromosome painting

Procedure of Chromosome Painting

Target DNA

Page 42: Ppt on karyotyping, chromosome banding and chromosome painting

ADVANTAGES OF FISH

RapidHigh efficiency of hybridization and

detectionLots of cells can be analyzed

Page 43: Ppt on karyotyping, chromosome banding and chromosome painting

Problems with in situ hybridization

Permeabilization problemsUneven cell penetration High amount of background

autofluorescene

Page 44: Ppt on karyotyping, chromosome banding and chromosome painting

Conclusion

. Studies structural features of each chromosomes.

Helps in studying Ideograms, chromosome banding pattern and Chromosomal painting techniques.

Helps in the identification of chromosomal aberrations.Helps in studying evolutionary changes.

Page 45: Ppt on karyotyping, chromosome banding and chromosome painting

REFERENCE

• Gupta ,P. K., 2012. Cytogenetics an advanced study, Chapter 1: 3-16.

• Wendy, A. B.,2001. Karyotype analysis and chromosome banding. Nature, 1-6.

• Moore, C. M. and Best, R. G., 2002. Chromosome preparation and banding. Nature, 1-6.

• Ried, T., et.al ,1998. Chromosome painting : a useful art. Human Molecular Genetics, Vol(7),1619- 1626.

Page 46: Ppt on karyotyping, chromosome banding and chromosome painting

THANK YOU