Karyotyping involves analyzing the morphology of chromosomes to characterize an organism's karyotype. Banding techniques like Q-, C-, G-, and R-banding involve treating chromosomes with dyes to distinguish different chromosome regions based on their DNA composition. In situ hybridization localizes specific DNA sequences on chromosomes using fluorescently-labeled probes. It is used for physical mapping, detecting chromosomal abnormalities, and studying phylogenetic relationships between species. Chromosome painting uses multiple color-coded probes to differentially label each chromosome pair for advanced analysis of structural variations.

1. KARYOTYPE

2. • The general morphology i.e., the size of
chromosome, the position of centromere, the
presence of secondary constrictions and the size of
satellite bodies of the somatic chromosome
complement of an individual constitutes its
karyotype.
• The karyotype of a normal somatic cell of a normal
individual represents the karyotype of the concerned
species.

3. • A perfectly symmetrical karyotype has all
metacentric chromosomes of the same size.
• Karyotype showing a deviation from this state
are called asymmetrical
• Species showing a greater asymmetry in their
karyotype are considered more advanced than
those showing less asymmetry.

4. IDIOTYPE
• The karyotype of a species may be repeated
diagrammatically showing all the morphological
features of the chromosomes.
• Gives same amount of the information as the
concerned karyotype.

5. APPLICATION OF KARYOTYPING
• To detect and study structural and numerical
chromosomal change.
• The karyotypes of different group compared and
similarities in karyotype are considered to represent
evolutionary relationship.
• It also suggests primitive or advanced feature of an
organism.

6. CHROMOSOME
BANDING

7. • Band is a part of chromosome which is clearly
distinguishable from its adjacent segments by appearing
darker or lighter with various banding methods.
• The artificial production of such bands by treatment with
specific dyes is referred to as chromosome banding.
• Banding techniques are based on identification of
chromosome segment that consist that consist of either AT
& GC rich region or of constitutive hetrochromatin.

8. • The pattern of chromosome banding is highly specific
in each chromosome of a species.
• Depending upon the pre treatment of the
chromosome and the used dyes or fluorochromes,
most common type of banding techniques are:
i. Q-banding
ii. C-banding
iii. G-banding
iv. R-banding

9. Q /Fluorescent
Banding
• First banding technique used by Caspersson, Zech
and Johannson(1970).
• Quinacrine mustard or quinacrine dihydrochloride
produces bright and dull flourescent bands known as
Q-Bands.
• Bright bands are composed of DNA that was rich in
the bases A&T due to formation of AT-quinacrin
complex fluorescens.

10. C-Banding
• Stains constitutive heterochromatin usually lies
near centromere.
• Heterochromatin binds a lot of the dye(Giemsa),
while the rest of the chromosomes absorb only
little of it.
Denaturation Renaturation Staining
• Well suited for the characterization of plant
chromosomes

11. G-Banding
• Uses geimsa dye.
- Giemsa stain, named after Gustav Giemsa,
- It is a mixture of methylene blue and eosin.
• No pretreatment before staining else resembles the C
Banding.
• G-Band visualizes the sulphur rich regions of chromosome.
• Well suited for animal cells.

12. R-Banding
• Stains regions rich in GC that are typical for euchromatin.
• Helpful in staining the distal ends of chromosome.
• Patterns reverse of G Bands.
• Helpful in staining the distal ends of chromosomes
therefore used to detect deletions and translocation that
involves telomeres of chromosomes
• Stained used- acridine orange dyes.

13. In situ
Hybridization

14. • in situ hybridization technique is to locate physical position of
a known DNA sequences on the chromosome, thus helps in
the physical mapping of genes or repeated DNA sequences.
• DNA is denatured then incubated in a solution of labelled
DNA, whose position on a chromosome, we are interested in
knowing
• Therefore repeated or unique DNA sequences can be utilized
as radioactive labelled or biotinylated probes for the study of
the location of these sequences on the chromosomes

15. General steps with minor variation
1. Label the probe (e.g. biotinylated UTP)
2. Preheat the slides and denaturate chromosomal DNA
3. Denature the probe and prepare hybridization mix
4. Place labelled probe and place the cover slip and seal and
incubate 4-14 hrs at 40°C
5. Remove cover slip and wash off hybridization mix.
6. Drain the slides but do not dry.
7. Subject to detection procedure(staining and visulization)

16. Fluorescencence
in situ
hybridization
• Used to detect the presence and absence of known
DNA sequence on chromosome.
• FISH involves the depositing a fluorescent molecule at
the site of in situ hybridization
• Fluorescence visualized under fluorescent microscope.
• Probes can be genome or chromosome specific DNA or
single copy sequence

17. STEPS INVOLVED IN FISH
Melting of DNA ( both in nucleus and in probe )
Labelling of probe with fluorescent molecule
eg.Biotin
Hybridization with the probe
Incubation in immuno fluorescent reagent
Fluorescent microscopy

18. ADVANTAGES
• High resolution
• High sensitivity and speed
• Variety of probe labelling schemes
• Hybridization of repeated sequence can be supressed
by prehybridization
• Both metaphase cells spread or interphase nuclei can
be fixed on slides.

19. GENOMIC IN SITU HYBRIDIZATION
• Total genomic DNA derived from alien species is used
as a probe, for in situ hybridization to identify the
presence of whole chromosome or chromosome
segment from the corresponding alien species in a
crop background.
• The probe is used together with excess amount of
unlabelled blocking DNA from the species being
probed, (also called genome blocking ).

20. • This excess DNA blocks the genome of species
being probed so that the labelled alien
genomic DNA may not hybridize with it, thus
permitting the detection of presence of the
alien chromatin.

21. Multi color FISH
• Used to detect the presence of introgressed
alien chromatin in a crop species.
• Uses mixtures of differently colored probes.
• Each chromosome identified by a characteristic color using whole
chromosome probe mixtures and variety of ratios of colors.
• Indirect method for mcFISH uses biotin or antibodies as
fluorescent molecules
• Direct method uses fluorochrome-labelled nucleotide for probe
labelling. Hence no need for immunocytochemical detection.

22. Application Of in situ Hybridization
• Chromosome mapping - utilized in many plants to
identify chromosome accurately using species specific
probes, ribosomal genes and even unique sequence.
• Genome analysis – GISH permits characterization of the
genome and chromosome of hybrid plants,allopolyploid
and recombinant inbred lines.

23. • Phylogenetic relationship – GISH offers new opportunities in
phylogenetic and taxonomic studies for determining and testing
genomic relationship of wild and cultivated plant species giving
unique information about similarities between DNA from
related species.
• Analysis of somaclonal variation – useful in identifying the type
of genomic changes that might occur during in vitro culture.
• Detection of alien chromatid – not only can be identified but
can also be counted in wide hybrids and amphidiploids.

24. • Detection of chromosomal abberations : it permits the
identification of small chromosome aberrations, which are
not readily detected by standard high resolution banding
technique.
• Chromosome organization at interphase nuclei : useful for
investigating chromosome organized in the interphase
nucleus, orientation of telomeres and centromeres, special
location of individual chromosome and the relationship
between chromatin decondensation and gene expression.

25. CHROMOSOME
PAINTING
• Refers to the hybridization of fluorescently labeled chromosome
specific, composite probe pools to cytological preparations
• The simultaneous hybridization of multiple chromosome painting
probes, each attached with a specific fluorochrome combination of
multiple chromosome painting probe, each tagged with a specific
fluorochrome resulting in differential color display of individual
chromosomes i.e., color karyotyping

26. APPLICATION OF CHROMOSOME
PAINTING
• Marker chromosome classification.
• Detection of small translocation which are
cytogenetically similar in appearance.
• To study complex chromosomal aberrations.

27. References
• Singh BD. 2009. Genetics. Kalyani Publications
• Devi J, Ko JM and Seo BB. 2005. FISH and GISH modern
cytogenetic techniques. Indian journal of
biotechnology. 4
• Gupta PK. 2007. Cytogenetics. Rastogi publications