This presentation is about the chromose structure, it's banding & painting. It includes the physical structure of chromosome, then karyotype & idiogram. Different types of chromosome banding & painting in details. FISH & GISH.

1. Presented By –
Shovan Das
B.Sc. (Agriculture)
Bidhan Chandra Krishi Viswavidyalaya

2. 
 Being a student of Genetics & Plant Breeding, I realised that a good
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information, but also a clear concept is important. I found there is not
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Still if you have any confusion or any suggestion please mail me at
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Preface

3. Chromosomes are -
• Rod-shaped, filamentous bodies present
in the nucleus
• Become visible during cell division
• Carriers of the genes or hereditary
materials
• Composed of thin chromatins which are
supercoiled during cell division
Structure of Chromosome
1. Chromatids: The longitudinal subunits of a chromosome are called
chromatids. These subunits of a chromosome get separated during anaphase.
Chromatids are of two types –
o Sister chromatids: Chromatids derived from the same chromosome.
o Non-sister chromatids: Chromatids originated from homologous
chromosome (not from same).

4. Chromonemata: Thread like coiled structures are found in chromatids, called
chromonemata (First observed by Baranetzky in 1880). It is the sub-unit of
chromatid. It has 3 main functions –
 Controls the size of chromosome.
 Helps in duplication of chromosome.
 Gene bearing portion of chromosome.
Chromonemata show two types of coiling –
• Paranemic: Chromonemal fibrils are simply lie alongside one anther &
easily separable from each other.
• Plectonomic: Chromonemal fibrils are closely intertwined and they cannot
be separated easily.

5. Chromomere: The linearly arranged bead like structures found on the
chromonemata is known as chromomeres (First described by Balbiani in
1876 and by Pfitzner in 1881). These are clearly visible in the polytene
chromosomes. It also has some functions –
 A unit of DNA replication.
 Chromosome coiling.
 RNA synthesis & RNA processing.
2. Primary Constriction or Centromere: A lighter stained (with Aceto-orcein,
Acetocarmine, Feulgen) region appears as a constriction or thinner segment
of the chromosome, called primary constriction or centromere. It has some
characteristics -
• Less densely coiled that’s why stained lightly by stains.
• This region consists of repetitive DNA sequence (constitutive
heterochromatin region)
• Have Cohesion Complex which holds sister chromatids together. Cohesion
complex is heterodimer of SMC (Structural maintenance of chromosome)
proteins (SMC1, SMC3). In this complex protein Scc1p released from the
sister chromatids to allow their separation at anaphase). Cohesion complex
is formed during S phase.

6. Centromeres are of two types –
o Point Centromeres: Point centromeres
are centromeres where mitotic spindle
fibres are attracted to specific sequences
of DNA with the help of some cell
proteins. Mitotic spindle fibres will
typically appear anywhere that the
DNA sequence of the point centromere
appears.
o Regional Centromeres: These are
centromeres where mitotic spindle
binding is determined, not by a precise
sequence of DNA, but by epigenetic
marks (reversible marks on DNA made
by enzymes without changing the
information contained in DNA).
Kinetochore: It is two discs of proteins (button like structure) located at
centromere on opposite sides on chromosome (one on each sister
chromatids). During cell division the microtubules/spindle fibres are
attached with this region.

7. 3. Secondary Constriction: The constricted or narrow region other than
that of centromere is called secondary constriction. It has some
characteristics –
• It has constant position and, therefore, can be used as useful marker.
• It is generally found on the short arm of a chromosome, away from the
centromere. But in some cases, it is located on the long arm.
NOR: Nucleolus is associated with this secondary constriction at
interphase, and it is originated at telophase stage of cell division. So that
secondary constriction is also known as Nucleolus Organizer Region
(NOR). This region is also constitutive heterochromatin and appears as a
light stained region.
Satellite: The region between secondary constriction and the nearest
telomere is called satellite. Generally some chromosomes contain satellite in
a genome; sometimes two satellites are present in a chromosome.
Chromosomes with satellites are called SAT (Sine Acido Thymonucleinico)
chromosome. In case of human chromosome no 13, 14, 15, 21 & 23 are SAT
chromosomes.

8. 4. Telomere: The terminal region of a chromosome on either side is known
as telomere. These are not visible in the light or electron microscope, they
are rather conceptual structures. Each chromosome has two telomeres.
Repetitive & palindromic sequences are found here. In human telomeric
sequence is TTAGGG. This region is also constitutive heterochromatin. Its
functions are –
 Prevents chromosomes to unite together by telomerase enzyme.
 Saves DNA from the activity of endonuclease enzyme.
 It is responsible for the aging of human as its length is shortening
gradually.
 Provides stability to chromosome.
5. Pellicle & Matrix: Each chromosome is bounded by a membrane called
pellicle. It is very thin and is formed of achromatic substance. This
membrane encloses a jelly-like substance which is usually called matrix. The
matrix is also formed of achromatic or nongenic material.

9. Karyotype & Ideogram
Karyotype: A karyotype is the identifying characteristics of number and
appearance of chromosomes, relative arm length, banding pattern,
centromere position and presence of satellite in decreasing order. Karyotype
is the stained chromosomes in metaphase stage of cell division.
Ideogram: An ideogram is a schematic diagrammatic representation of
the karyotype that shows all of the pairs of homologous chromosomes in
the nucleus. The pairs of chromosomes are lined up in order of size, so
that the centromeres are aligned and the short arm is uppermost. It is
drawing of sets of chromosome, not the actual picture of chromosome.

10. Karyotyping is done by staining chromosomes and so on
different band is observed on chromosome. The euchromatin
regions of chromosome are stained deeply and the light
stained regions are heterochromatin.
• In a chromosome, a centromere which divides each
chromosome into long and short arms. The short arm is
denoted by the letter p (petit) and the long arm by the
letter q (queue).
• For example, the short arm of chromosome 5 is denoted
simply by writing “5p.” Thus, in the short arm of
chromosome 5, we have region 5p11, which is closest to
the centromere, followed by regions 5p12, 5p13, 5p14, and
5p15, which is farthest from the centromere.

11. •Within each region, individual bands are denoted by numbers following a
decimal point; for example, 13.1, 13.2, and 13.3 refer to the three bands that
make up region 5p13. The pattern of bands within the chromosome is called
an idiogram.
Advantages of Karyotype:
• Reveals the structural features of each chromosome in a genome.
• Helps in identifying chromosomal aberrations.
• Helps in studying chromosome binding pattern.
• Detection of prenatal genetic disorders.
• Helps in study of evolutionary changes.
• Detection of specific chromosome in plants.
Disadvantages of Karyotype:
• Very small abnormality cannot be shown by karyotyping.
• An unknown chromosome cannot be identified by karyotyping.

12. Chromosome Banding
Chromosome band: Transverse
bands produced on chromosomes
by differential staining
techniques. Depending on the
particular staining technique, the
bands are alternating light and
dark. Each human chromosome
has a short arm ("p" for "petit")
and long arm ("q" for "queue")
separated by a centromere. The
ends of the chromosome are
called telomeres. The ends of the
chromosomes are labelled ptel
and qtel. For example, the
notation 7qtel refers to the
telomere (the end) of the long arm
of chromosome 7.

13. Q-Banding
Discovered by: Caspersson et. Al (1958)
Procedure:
Observation: The AT rich regions (Heterochromatin regions) produces dark strain & GC rich regions (Euchromatin
regions) produces light strain)
Advantages:
1. It is a simple and versatile technique.
2. It is used as a method of identifying chromosomes in combination with other procedure.
3. Study of heteromorphism.
4. Study of human Y chromosome.
Disadvantages:
1. The tendency to fade during examination.
2. Photo degradation.
3. Chromophores absorb a particular wavelength light due to a chemical bond formed between dye & light.
4. UV light breaks the chemical bonds.
Chromosome
Stained with Quinarcine Mustard
UV Light
Banding Pattern

14. G-Banding
Discovered by: Summer et. Al (1971)
Procedure:
Observation: The AT regions (sulphur rich) stained dark and the GC regions stained lightly. This method will normally
produce 400–600 bands in a normal human genome in metaphase chromosome.
Advantages:
1. It is used for demonstrating euchromatic bands.
2. Identification of sulphur rich regions of chromosome.
3. Identification of chromosomal abnormalities.
4. Gene mapping & high resolution banding.
Disadvantages:
1. Ineffective of determining small translocations & microdeletions.
2. Can’t characterize the chromosomes of complex cell lines.
3. Not used in plants.
Why G-Banding technique isn’t used in plants?
1. Plant chromosomes in metaphase contain much more DNA than G-banding vertebrate chromosomes of
comparable length. So plant chromosomes wouldn’t show band for optical issue.
2. Vertebrate metaphase chromosomes are 2.3 times shorter that of pachytene, but plant metaphase chromosomes are
10 times shorter than of pachytene. Hence it is difficult to demonstrate the arrangement of bands.
Chromosome
Treat with Trypsin/Urea/Protiase for
denaturation
Treat with
Giemsa
Banding Pattern

15. R-Banding
Procedure:
Observation: It is the reverse technique of G-Banding. Here the GC rich euchromatin regions
stained dark than AT regions. The salt solution denatures the DNA of AT rich regions, so that
that region stained lightly.
Advantages:
1. Helpful for analysing the structures of chromosome ends (telomeres).
2. Used to identify the euchromatin regions of chromosome.
Chromosome
Treated with
Salt Solution
Treat with
Giemsa
Banding
Pattern

16. N-Banding
Discovered by: Matsui & Sasaki (1973)
Procedure:
Observation: Dark stained band found at secondary constriction, NOR region, satellite etc.
Advantages:
1. Used for the identification of NOR.
2. Superior banding patterns for plants.
Disadvantages:
1. Number of bands is less in compare to NOR staining.
2. Bands don’t retain for long time.
Chromosome
Air Dried & stained with Giemsa (with phosphate
buffer)
Treated with 5% Trichloroacetic Acid at 95⁰C
for 30 min
Treated with 0.1N HCL at 60⁰C for 30
min
Banding Pattern

17. C-Banding
Discovered by: Linde & Laursen
Procedure:
Observation: Bands found at constitutive heterochromatin region i.e. in centromere region
specially.
Advantages:
1. Identification of insect and plant chromosomes.
2. Identification of bivalents at diakinesis using both centromere position.
3. Paternity testing.
4. Gene mapping.
Chromosome
Treated with alkali solution
Washing with sodium citrate at 60⁰C for 30 min
Staining with Giemsa Solution
Banding pattern

18. T-Banding
T-Banding is also used for identifying the telomeric regions. It was first
reported by Dutrillaux. In this case controlled thermal denaturation of
chromosome is done followed by staining with Giemsa or acridine orange.
The T-bands apparently represents the subsets of R-bands as they are
smaller compared to R-bands.
Hy-Banding
This is a common technique used in plant cells. Cells are pre-treated with
HCl and stained with acetocarmine. Banding found in histone rich regions
NOR-Staining
Chromosomes are treated with silver nitrate solution which binds to NOR
region. This staining is quite advanced to the Giemsa N-banding. Because
the number of bands in silver staining are more than N-banding. Also
silver stained slides lasts more days than N-banding (even N-banded
slides don’t last for 1 week).

19. Chromosome Painting
Chromosome painting involves the use of fluorescent-tagged chromosome
specific DNA sequences to visualize specific chromosomes or chromosome
segments by in situ DNA hybridization and fluorescence microscopy.
Chromosome painting refers to the hybridization of fluorescently labelled
chromosome-specific, composite probes to cytological preparations.
Importance of chromosome painting:
• Improves the efficiency of screening cells for chromosome abnormalities.
• The understanding of chromosome changes that occurred during the
evolution of species.
• Homologies between the chromosomes of different species can be
detected by chromosome painting.
• In recent years, the complete karyotypes of various mammals including
primates, carnivores and artiodactyls have been analysed by
chromosome painting.
• Used to identify reciprocal translocations which are difficult to identify through
simple staining.

20. About Chromosome Painting:
• Chromosome Painting – the term first used by Pinkel et. Al in 1988.
• Chromosome painting was developed independently by research
teams at Lawrence Livermore National Laboratories and at Yale
University.
• In-Situ Hybridization technique was developed by Joseph G Gall,
Mary Iou Pardue and John et. al in 1969.
• FISH was first applied to plant cytogenetics by Leitch et. al in 1991.
Fluorescence in situ hybridization (FISH):
Fluorescence in situ hybridization (FISH) is a molecular
cytogenetic technique that uses fluorescent probes that bind to only those
parts of a nucleic acid sequence with a high degree of
sequence complementarity.

21. Procedure of FISH:
Flow sorted
chromosomes(Probe)
Amplification by PCR
Tagged with fluorophores
Denature the probes at 65⁰C
for 10 mins
Prepare slides with metaphase
chromosomes (target)
Dehydrate (in ethanol) &
denature in 70⁰C of metaphase
fixed chromosomes
Incubate probe & unlabelled
DNA at 37⁰C for 4-16 hours
Wash & detect with fluorocent
microscope

22. Types of probes used in FISH:
1. Locus specific probes: Bind to
particular region of chromosome,
used for specific gene
identification.
2. Centromeric or Alphoid probes:
Made from repetitive sequences,
used for searching missing part of
genes.
3. Whole chromosome probes:
Collection of smaller probes
which binds with target
chromosome along the length.
Types of FISH:
• Stellaris RNA FISH: It is a single molecule RNA FISH, detects &
quantifies the m-RNA & other long RNA in tissues.
• Fiber FISH: FISH technique by attaching chromosomes linearly stretched
out on slides.
• Q-FISH: Combines peptide nucleic acid (PNA) & computer software,
used for telomere length research.

23. • Flow-FISH: Flow-FISH uses flow cytometry to perform FISH
automatically using per-cell fluorescence measurements.
• MA-FISH: Microfluidics-assisted FISH uses a microfluidic flow to increase
DNA hybridization efficiency, applied for detecting the HER2 gene in
breast cancer tissues.
• Hybrid Fusion-FISH: Hybrid Fusion FISH uses primary additive
excitation/emission combination of fluorophores to generate additional
spectra (total 7 detectable emission spectra) through a labelling process
known as dynamic optical transmission (DOT). Hybrid Fusion FISH
enables highly multiplexed FISH applications that are targeted within
clinical oncology panels.
• FS-FISH: Fusion signal FISH used for detecting Philadelphia
translocations.
• Immuno-FISH: Immuno-labelling with FISH for detecting proteins &
specific DNA sequences in chromosomes.
• M-FISH: Multicolour FISH is the 24 colour karyotyping.
• ML-FISH: Multilocus FISH used for screening of multiple microdeletions.
• LNA-FISH: Locked nucleic acid FISH.
• Cryo-FISH: Cryosection FISH.
• D-FISH: Double fusion FISH.
• COD-FISH: Chromosomal orientation & direction FISH.
• ACM-FISH: Alpha (centromeric), classical and midi satellite FISH.
• CAT-FISH: Cellular compartment annalists of temporal activity FISH.

24. Advantages of FISH:
• Rapid method.
• Efficiency is high.
• Sensitivity & specificity is high.
• Data can be obtained from poor
samples.
• The method is automatic system.
Limitations of FISH:
• Abnormalities can be detected with
currently available probes.
• One or few abnormalities can be
assessed simultaneously.
• High sensitive for trisomy but less
sensitive for deletions.
• Requires fluorescence microscopy &
image analysing system.
• Permeability problems for probes.
• High amount of background
fluorescence.
FISH analysis of the LEF1 locus in human
colon cancer cell

25. Genomic in situ hybridization (GISH):
It is the same process as FISH but an entire genome is used as a probe in
this case. GISH is an absolute approach, used for establishing phylogenetic
relationship.
Forward Painting & Reverse Painting
Forward Painting: In forward painting probes are prepared from the
normal genome sets & used for hybridization. In this process fluorescence
can be detected from all labelled DNA, aberrations/rearrangements can be
found in the basis of abnormal fluorescence. But in this case chromosomal
abnormalities can’t be found (or hard to find) in case of unknown
chromosomes/genotypes. This is the conventional/traditional method of
chromosome painting having some limitations.
Reverse Painting: In this case probes are prepared from aberrant
chromosomes of any genome. So naturally probes attach with the
aberrant/abnormal chromosomes and produce fluorescence. Probes are
prepared by degenerate oligo-nucleotide prime PCR (DOP-PCR). As
example, a primer 6MW have sequence - 5’ CCG ACT CGA GNN NNN
NAT GTG G 3’; where N= any base.

26. Reference:
• Cytogenetic Analysis by Chromosome Painting – Nigel P. Carter,
University of Cambridge, Cambridge, United Kingdom
• Chromosome Painting – Sharma A. K.
• Genetics – B.D. Singh
Source:
• Wikipedia
• www.biologydiscussion.com
• www.biologydictionary.net
• www.dna-rainbow.org
• Frontiers in Plant Science
• www.study.com
• www.iaszoology.com
• BMC Plant Biology
• www.nature.com
• www.springer.com
• Oxford Academic