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WELCOME
Karyotyping, Chromosome Banding and Chromosome Painting
1.Karyotyping
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
• 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.
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”.
Types of Karyotype
Karyotype
Symmetric Karyotype
Asymmetric Karyotype
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
• 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.
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
Procedure of karyotyping
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
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
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 .
2.Chromosome Banding
Introduction
History
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.
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.
Banding Techniques
Q(Quinarcine)
G (Giemsa) N (NOR)
C(Centromeric)
1958 1971 1973 1978
Casperson et.al
Summer et.al
Matsui & Sasaki
Linde &Laursen
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
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.
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
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.
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.
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
N Banding Techniques
Advantages• Used in the identification of Nucleolar
organizer region.• Superior banding pattern for plants.
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
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.
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.
• 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.
• 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.
3.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.
WHY?????
Helps in the identification of Chromosomal rearrangements.
Helps in the identification of Chromosomal breakpoints.
Helps in determination of extra chromosomal material.
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.
Procedure of Chromosome Painting
Target DNA
ADVANTAGES OF FISH
RapidHigh efficiency of hybridization and
detectionLots of cells can be analyzed
Problems with in situ hybridization
Permeabilization problemsUneven cell penetration High amount of background
autofluorescene
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.
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.
THANK YOU