ST XAVIER’S COLLEGE RANCHI

TOPIC: CHROMOSOME BANDING,CHROMOSE

MAPPING,SEM AND TEM
SUBJECT: ANALYTICAL TECHNIQUES IN PLANT
SCIENCES

NAME: SWETA SHALINI

CLASS ROLL NO:647
EXAM ROLL NO:15SBOT027612
PAPER :DISCIPLINE SPECIFIC
ELECTIVE
 CERTIFICATE

This project is to certify that this project has been made by

SWETASHALINI OF 3RD year on the topic inbreeding depression and heterosis
under the guidance of our discipline specific elective paper professor ARSHI
NAAZ has been completed successfully under the session 2015-2018.

DATE: SIGNATURE:
 ACKNOWLEDGMENT

FIRST of all I would like to thank god for providing me

strength to complete this project with all strength.
Secondly I would like to thank my principal father
for providing this opportunity of this project.
I would like to thank my teacher Arshi naaz for
providing us ample of time and suggestions whenever needed.
At last but not the least I would like to thank my
family for helping me both finically and physically whenever
needed.
I hope all mistakes will be highly acknowledged.
 CONTENT
Chromosome painting introduction TEM
•Requisites and labelling methods APPLICATIONS
•Different strategies HISTORY
•Application and procedure PROCEDURE
Advantages and disadvantages
Fish CHROMOSOME BANDING
Procedure Introduction
Applications G banding
History C banding
Advantages and disadvantages R banding
Q banding
Types  CONCLUSION
Procedure
How does it works
 SEM
HISTORY
APPLICATIONS
ADVANTAGES AND Disadvantage
Preparation
 INTRODUCTION
Chromosome Painting is emerging as a powerful tool in the exact localization of different gene
sequences of chromosomes at the microscopic level. It is principally based on molecular
hybridization in situ with sequence specific probes on chromosomes. Different strategies have
been adoptedfor the preparation of probes, hybridization and visualization. The impact of this
method lies in identification of genes for desired characters in the chromosomes, including
those of genetic disorders, in cancer research, in transgenesis and in studies on biodiversity
and evolution.
The term Chromosome Painting widely implies painting of differential chromosome segments
with sequence specific probes, and is principally based on the technique of in situ molecular
hybridization. The term in the strict sense, however, was used earlier along with chromosomal
in situ suppression (CISS) [14, 18] of dispersed repeats in human system, for in situ labelling of
chromosome or chromosome regions, with segments unique to them. It is essential both for
structural and functional genomics. Introduction The localization and mapping of functionally
differentiated segments of chromosomes and gene loci have been greatly facilitated through
the application of molecular hybridization technique at the chromosome level.
 The knowledge of the structural complexity of chromosomes at the molecular level, and
the sequence complexity, has been gathered through an analysis of annealing of
complementary sequences following hybridization of RNA and DNA strands. The method of
in situ molecular hybridization principally uses probe sequences tagged with radioisotopes
or a chemical reporter. The initial step is denaturation of the target chromosomal DNA
mostly in metaphase, to facilitate access of the probe to the target. This is followed by
hybridization with the probe, while complementary sequences undergo pairing. The
hybridized sites are localized either through autoradiography or immunofluorescence,
depending on the type of probe used [see 13], as well as counter with specific stains for
the preparation of probes, hybridization and visualization. The impact of this method lies
in identification of genes for desired characters in the chromosomes, including those of
genetic disorders, in cancer research, in transgenesis and in studies on biodiversity and
evolution.In cases where specific species propes are species across taxonomic barrier, the
term comparative chromosome painting or cross species chromosome painting CCCP or
Zoo FISH [12] is applied as against normal chromosome painting, where the application is
on its own genome. Of the two types of molecular hybridization, isotopic and non-isotopic,
the chromosome painting is based on the non-isotopic one, the underlying principle of
which is to make the specific loci of DNA antigenic [6]. Discovery of various chemical
reporter molecules, which can be tagged with the probes, and final detection through the
appropriate fluorescing compound, has become a fascinating technique, widely pursued in
various centres.
 REQUISITES
Analysis of the different gene blocks in chromosomes
and segments of complex genome has been carried out through the development and use
of probes with differential fluorescence. Such multicolour techniques have been used to
localise repeated DNA and other sequences on chromosomes [10, 18]. The method of
multicolour preparations in chromosome painting may be direct or indirect.

LABELLING METHODS
Indirect labelling of probes involves the use of
compounds such as biotin as the primary labels and later streptavidins, which are used as
a conjugate for signal generation system. Otherwise, antibody of a hapten is incorporated
into the probe, the recognition of which leads to detection. Similarly, photobiotin is
detected either through luminescence or fluorescence or alkaline phosphatase conjugate
– colorimetrically with enzyme conjugates. In general, the indirect methods use probes
tagged with biotin, digoxigenin and dinitrophenol (DNP) as reporter molecules, which are
detected by fluorochrome-conjugated avidin or antibodies. The common fluorochromes
are FITC (fluorescin-isothiocyanate), rhodamine and AMCA (amino-r-methyl coumarin-
3acetic acid). Isomer of biotin (mol. wt. 244.31) is a natural vitamin of the animal cell,
pecially of liver, kidney and pancreas. It has a few chromophoric groups .
Advin is whereas a tetrametic group animal glyco-protein and all four tetramers have four
biotin binding sites. The avidin-biotin interaction is the strongest noncovalent biological
recognition so far known between ligand and protein. The reaction is rather rapid; not
affected by pH, solvents or denaturing agents and can withstand short exposure to high
temperatures more than 100 °C. Avidin denaturation may, however, affect the binding
capacity [15]. The long immunological detection method can be avoided by using
fluorescin-12-dUTP instead of dinitrophenol as reporter. The direct method on the other
hand, is characterized by probe labelling with fluorochrome-labelled antibodies. In direct
labelling, the signal generation system is directly attached to the probe which is detected
post-hybridization with enzyme and enzyme conjugate. The examples include horse
radish peroxidase where the detection is through high chemo luminescence. Such direct
coupling of fluorochromes to probes does not need detection through
immunocytochemical methods. The direct labelling of fluorochrome into the probe is
rapid and ensures good resolution. Probes for single, large and repetitive resolution

PROBES FOR SINGLE LARGE REPETITIVE SEQUENCES

For detection of hybridization, the nature of the probe is an important factor – the larger
the probe, the easier is the detection. However, with the gradual development of
amplification method at in situ level, even single copy sequences are amenable to
detection [13]. Probes for chromosome painting can be obtained either by (i) flow sorting
or from (ii) microdissection of chromosomes.
The use of flow sorting has been widely used in animal system as compared to microdissection. For complex larger
probes, biotin labels are preferred, in view of the convenience of immunocytochemical detection of this compound.
The objective underlying labelling is to incorporate biotinylated analogue of dUTP into probe by nick translation.
Biotin is linked by a linker to C5 position of the pyramiding. With short linkers, the linkage is strong, whereas with long
linkers, their detection through detector is more efficient. Probes of chromosome painting can be obtained directly
either by
(i) flow sorting or from
(ii) micro dissection of chromosomes.
The flow sorting has been widely used in the animal system as compared to microdissection. For complex larger
probes, biotin labels are preferred, in view of the convenience of immunocytochemical detection of this compound.
The objective underlying labelling is to incorporate biotinylated analogue of dUTP into probe by nick translation.
Biotin is linked by a linker to C5 position of the pyrimidine. With short linkers, the linkage is strong, whereas with long
linkers, their detection through detector is more efficient hybridization sites is done by application of avidin,
conjugated with fluorochrome or antibiotin antibodies. For single copy probes, biotinylated anti-avidin
antibodies, followed by avidin fluorophore isothiocynate or rhodamine, such as fluorescin, are
generally used. Counter-staining with propidium iodide or DAPI is preferred. Various techniques can
also be adopted after detection of the probe signal. Observations are carried out under fluorescent
microscope with different combinations of emission and excitation filters. The repetitive sequences
often hamper the identification of complex DNA sequences. Because of their non-specificity, these
sequences hybridize with their corresponding sequences present in the genome. In order to overcome
this problem, chromosomal in situ suppression (CISS) or competitive in situ hybridization (CISH) is
adopted. This method involves preannealing the probe with appropriate competitor DNA, rendering
the reptitive sequences, specially the dispersed sequences, unavailable for hybridization. Normally, the
competitive DNA is the total fragmented unlabelled genomic DNA, enriched with repetitive sequences
of desired length. The probe can then be amplified through polymerase chain reaction.
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.
Chromosome painting allows the visualization of individual chromosomes in metaphase or
interphase stages and the identification of both numerical and structural chromosomal aberrations
with high sensitivity and specificity. The siimultaneous hybridization of multiple chromosome
painting probes, each tagged with a specific light-emitting fluorochrome has resulted in the
differential colour display of human and mouse chromosomes, which is also called colour
karyotyping.
Fluorescent in situ hybridization (FISH) has been used to detect the location of specific genomic
targets using probes that are labelled with specific fluorochromes. That is the reason chromosome
painting is also called M-FISH or multicolour FISH. The technique allowed detection of simple and
complex chromosomal rearrangements. In addition, complex chromosomal abnormalities could also
be identified that could not be detected by the conventional cytogenetic banding techniques.
Almost a decade ago, chromosome painting was developed independently by research teams at
Lawrence Livermore National Laboratories and at Yale University. Both groups had taken advantage
of the availability of cloned DNA libraries that were derived from flow-sorted human chromosomes.
The first generation of probes, based on chromosome-specific phage libraries, were rather
cumbersome to use, due to low insert-to-vector ratios, which frequently resulted in a relatively high
background staining. Some of these limitations were overcome with the availability of plasmid
libraries, where an improved insert-to-vector.
•Chromosome painting refers to the hybridisation of fluorescence labelled chromosomes
specific, composite probe pools to cytological preparations
•It was first named by PINKEL et.al in 1988
•Chromosome painting coupled with fluorescence in situ hybridization is used routinely for
identification of chromosomes.

WHY chromosal painting?????

Helps in identification of chromosal rearrangements.
Helps in identification of chromosal breakpoints.
Helps in determination of extra chromosal material.
 DIFFERENT STRATEGIES

Different methods have so far been developed to delineate sequences specific to

chromosome, chromosome region or even genome.

FISH AND GFISH

The initial approach of fluorescence in situ hybrfinements. However, FISH technique
provided the possibility of utilizing even large or comparatively shorter sequences using
bacterial artificial chromosome (BAC) or idization (FISH) with the sequences, regions or
chromosomes as probe and later, the in situ hybridization with total genome (GISH) as
probe laid the foundation for further reyeast artificial chromosomes (YAC) as probes.
Mammalian system in general, including man, with comparatively low genome contents,
yielded excellent results in chromsome painting. Plants with large complex genome with
similarity of repeats initially presented difficulties in delineating desired repeated
sequences with specificity, as compared to animal system, where painting of short
unique sequences is carried out, along with chromosomal in situ suppression of fast
reassociating repeats. The DNA probes derived from chromosomes of specific regions of
chromosomes as in Vicia faba (field bean) or Picea abies (spruce) led to the labelling of
almost all chromosome regions.
Chromosome painting has improved the efficiency of screening cells for chromosome
abnormalities, in testing chemicals for mutagenacity and for rearrangements associated
with tumours. Painting probes detect chromosome rearrangements. Use of the same
chromosome paints for chromosomes of different species reveals the extent of
chromosome rearrangements since divergence of the species.
Chromosome painting probes are now also available for an ever increasing number of
species, most notably for the mouse and the rat, allowing the expansion of chromosome
painting analyses to animal models for human diseases. FISH techniques have been
developed and applied to identify the origin of the markers and other structural
chromosomal aberrations. The use of chromosome painting probes in one, two or three
colour FISH experiments has significantly improved the definitive diagnosis of chromosomal
aberrations.
The introduction of chromosome painting to the field of comparative cytogenetics has
added significantly to the understanding of chromosome changes that occurred during the
evolution of species. Chromosome painting can be used to identify homologous
chromosome segments in different species and to map probes of different complexities and
chromosome rearrangements in a single experiment. In recent years, the complete
karyotypes of various mammals including primates, carnivores and artiodactyls have been
analyzed by chromosome painting.
Chromosome Painting Probes, available in liquid format, are directly labelled in either a red
or green fluorophore. They can be mixed together to label a number of chromosomes in a
single reaction. The probes come in the form of ready to use hybridization solution in a five-
test kit format, and are supplied complete with DAPI counter stain. The protocol is rapid
and simple and follows simultaneous co-denaturation of the FISH probe and target DNA.
 The origin of marker chromosomes that were unidentifiable by standard banding
techniques could be verified by reverse chromosome painting. This technique includes
micro-dissection, followed by in vitroDNA amplification and fluorescence in
situ hybridization (FISH). The chromosomal material is amplified by a degenerate
oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products
are labelled by nick-translation with biotin-11-dUTP and used as probes for FISH.
Homologies between the chromosomes of different species can
be detected by chromosome painting. A more detailed description of how this method of
chromosome painting works is given below:
•· Initially suspensions of chromosomes from dividing cells are sorted by a method known as
flow cytometry.
•· DNA from one chromosome is then labelled with a fluorescent dye using a technique
called fluorescence in situ hybridisation (FISH). This labelled DNA paints the chromosome
and allows homologous regions of DNA of other species, from great apes to mice, to be
identified. Homologous regions show up in the same colour on the chromosome charts.
•· If you take a paint made from human chromosome 2 and hybridise it onto the
chromosomes of another species, then you’ll see segments of paint on different
chromosomes, each being homologous to part of human chromosome 2, so with the gibbon
karyotype, regions on chromosome 2 in human can be tracked to 5 different chromosomes
in gibbon. The fluorescent-labelled DNAs will attach to the analogous chromosomes from
which they were derived. DNA fragments with the same base sequences have the
characteristic of attaching to each other.
•· This tells us that the lineage that produced gibbon and the lineage that produced human
have diverged over several million years and during this time rearrangements have occurred
between the two species which can be tracked using the painting technique.
• If a part of a painted chromosome (yellow, for example) had undergone an exchange
with another, non-painted chromosomes (stained red), it is possible to detect the
aberration.
• Usually, a pair of bi-coloured chromosomes can be detected in one metaphase, because
two chromosomes typically exchange a part of their DNA. Reciprocal translocations are
difficult to detect by simple staining technique that stains the entire set of chromosomes
with a single material such as with Giemsa.
• · When human chromosome probes are hybridised to the chromosomes from other
species, the same set of blocks, dispersed across multiple human chromosomes, are
often located together on one chromosome in other species. For example, parts of
human chromosome 3 and 21, or 14 and 15, or 12 and 22, tend to be located together
on one chromosome in other animals. This is evidence that an ancestral block of genes
has been split apart and moved to different chromosomes during human evolution.
• · Collating all these patterns of linked regions of chromosome across various mammalian
species has enabled to assemble a picture of genomic commonalities across multiple
species.
 APPLICATIONS

To find out the location of a specific gene located on a specific chromosome: FISH
hybridization is done with the appropriate gene-specific probe labelled with the
fluorescent dye. The test will give the binding of the gene-specific probe labelled with
the fluorescent dye to the respective chromosome at the specific position, where the
gene is located.
Detection of translocation: Chromosomes that have undergone translocation will have
two segments. When it is subjected to the technique of chromosome painting it will
take different probes and appear in two colours or multicoloured depending on the
number of translocations.
Detecting chromosome abnormalities: FISH has improved the efficiency of
screening cells for chromosome abnormalities in mutagenic studies and for testing
the mutagenic ability of chemicals and other potent mutagens in the environment.
It has also improved the detection of chromosome aberrations and rearrangements
associated with tumour and cancer.
 PROCEDURE SAMPLE PREPARATION
AND HYBRIDISATION

Prepare slides with metaphase chromosomes

Dehydrate in ethanol
Denature DNAat70c
Denature labelled probe
Incubate at 37c for 4-16hours for hybridization
 FISH
This technique was developed in the late 1980s and is a powerful method to detect translocations
(rearrangements among chromosomes).For the development of FISH, it was necessary to isolate
each human chromosome. Subsequently, DNA from these chromosomes was fragmented and put
into bacterial cells to amplify it (produce many copies). In this way, a large number of copies of DNA
from each chromosome can be obtained.
These amplified DNA fragments are labelled with appropriate fluorescent (light-emitting)
dyes and allowed to hybridize (attach) to metaphase chromosomes. The fluorescent-labeled DNAs
will attach to the analogous chromosomes from which they were derived. (DNA fragments with the
same base sequences have the characteristic of attaching to each other.)
In this way, if a part of a painted chromosome (yellow, for example) had undergone an
exchange with another, non-painted chromosomes (stained red), it is possible to detect the
aberration (termed a reciprocal translocation) because the aberrant chromosome contains both
yellow and red segments. Usually, a pair of bi-coloured chromosomes can be detected in one
metaphase because two chromosomes typically exchange a part of their DNA. Reciprocal
translocations are difficult to detect by simple staining the entire set of chromosomes with a single
material, such as with Giemsa. Imagine, for example, a case with which the two exchanged
segments had similar lengths. The two translocated chromosomes should appear perfectly normal,
both in shape and length. If we employ FISH, however, such translocation can be clearly detected.
Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent
probes that bind to only those parts of the chromosome with a high degree of
sequence complementarity. It was developed by biomedical researchers in the early 1980 and 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.FISH can also be used to detect and localize specific RNA targets
(mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples. In this context, it can
help define the spatial-temporal patterns of gene expression within cells and tissues. Probes – RNA and
DNA.
RNA probes can be designed for any gene or any sequence within a gene for visualization
of mRNA, lncRNA and miRNA in tissues and cells. FISH is used by examining the cellular reproduction
cycle, specifically interphase of the nuclei for any chromosomal abnormalities. FISH allows the analysis
of a large series of archival cases much easier to identify the pinpointed chromosome by creating a
probe with an artificial chromosomal foundation that will attract similar chromosomes. The
hybridization signals for each probe when a nucleic abnormality is detected. Each probe for the
detection of mRNA and lncRNA is composed of 20 oligonucleotide pairs, each pair covering a space of
40–50 bp. For miRNA detection, the probes use proprietary chemistry for specific detection of miRNA
and cover the entire miRNA sequence. Urothelial cells marked with four different probes.
Probes are often derived from fragments of DNA that were isolated, purified, and
amplified for use in the Human Genome Project. The size of the human genome is so large, compared to
the length that could be sequenced directly, that it was necessary to divide the genome into fragments.
 PREPARATION AND HYBRIDIZATION
PROCESS
Cells, circulating tumor cells (CTCs), or formalin-fixed paraffin-embedded (FFPE) or frozen
tissue sections are fixed, then permeabilized to allow target accessibility. FISH has also
been successfully done on unfixed cells . A target-specific probe, composed of 20
oligonucleotide pairs, hybridizes to the target RNA(s). Separate but compatible signal
amplification systems enable the multiplex assay (up to two targets per assay). Signal
amplification is achieved via series of sequential hybridization steps. At the end of the
assay the tissue samples are visualized under a fluorescence microscope.
Scheme of the principle of the FISH Experiment to localize a gene in the nucleus.
First, a probe is constructed. The probe must be large enough to hybridize specifically with
its target but not so large as to impede the hybridization process. The probe
is tagged directly with fluorophores, with targets for antibodies or with biotin. Tagging can
be done in various ways, such as nick translation, or PCR using tagged nucleotides.
Then, an interphase or metaphase chromosome preparation is produced. The
chromosomes are firmly attached to a substrate, usually glass. Repetitive DNA sequences
must be blocked by adding short fragments of DNA to the sample. The probe is then
applied to the chromosome DNA and incubated for approximately 12 hours while
hybridizing. Several wash steps remove all unhybridized or partially hybridized probes.
Single-molecule RNA FISH
It is also known as Stellaris RNA FISH, is a method of detecting and quantifying mRNA and
other long RNA molecules in a thin layer of tissue sample. Targets can be reliably imaged
through the application of multiple short singly labelled oligonucleotide probes. The binding
of up to 48 fluorescent labelled oligos to a single molecule of mRNA provides sufficient
fluorescence to accurately detect and localize each target mRNA in a wide-field fluorescent
microscopy image.
Fiber FISH
In an alternative technique to interphase or metaphase preparations, fiber FISH,
interphase chromosomes are attached to a slide in such a way that they are stretched out
in a straight line, rather than being tightly coiled, as in conventional FISH, or adopting
a chromosome territory conformation, as in interphase FISH
Q-FISH
Q-FISH combines FISH with PNAs and computer software to quantify fluorescence
intensity. This technique is used routinely in telomere length research.
Flow-FISH
Flow-FISH uses flow cytometry to perform FISH automatically using per-cell fluorescence
measurements.
MA-FISH
Microfluidics-assisted FISH (MA-FISH) uses a microfluidic flow to increase DNA hybridization
efficiency, decreasing expensive FISH probe consumption and reduce the hybridization time.
 MEDICAL APPLICATIONS
Often parents of children with a developmental disability want to know more about their
child's conditions before choosing to have another child. These concerns can be addressed
by analysis of the parents' and child's DNA. In cases where the child's developmental
disability is not understood, the cause of it can potentially be determined using FISH
and cytogenetic techniques. Examples of diseases that are diagnosed using FISH
include Prader-Willi syndrome, Angelman syndrome, 22q13 deletion syndrome, chronic
myelogenous leukemia,acute lymphoblastic leukemia, Cri-du-chat, Velocardiofacial
syndrome, and Down syndrome. FISH on sperm cells is indicated for men with an abnormal
somatic or meiotic karyotype as well as those with oligozoospermia, since approximately
50% of oligozoospermic men have an increased rate of sperm chromosome abnormalities.
The analysis of chromosomes 21, X, and Y is enough to identify oligozoospermic individuals
at FISH can be used to form a diagnosis, to evaluate prognosis, or to evaluate remission of
a disease, such as cancer. Treatment can then be specifically tailored. A traditional exam
involving metaphase chromosome analysis is often unable to identify features that
distinguish one disease from another, due to subtle chromosomal features; FISH can
elucidate these differences. FISH can also be used to detect diseased cells more easily than
standard Cytogenetic methods, which require dividing cells and requires labor and time-
intensive manual preparation and analysis of the slides by a technologist.
Species identification
FISH is often used in clinical studies. If a patient is infected with a suspected pathogen,
bacteria, from the patient's tissues or fluids, are typically grown on agar to determine the
identity of the pathogen. Many bacteria, however, even well-known species, do not grow
well under laboratory conditions. FISH can be used to detect directly the presence of the
suspect on small samples of patient's tissue.
FISH can also be used to compare the genomes of two biological species, to
deduce evolutionary relationships. A similar hybridization technique is called a zoo blot.
Bacterial FISH probes are often primers for the 16s rRNA region.
FISH is widely used in the field of microbial ecology, to identify microorganisms. Biofilms,
for example, are composed of complex (often) multi-species bacterial organizations.
Comparative genomic hybridization
Comparative genomic hybridization can be described as a method that uses FISH in a
parallel manner with the comparison of the hybridization strength to recall any major
disruptions in the duplication process of the DNA sequences in the genome of the nucleus.
Virtual karyotype
Virtual karyotyping is another cost-effective, clinically available alternative to FISH panels
using thousands to millions of probes on a single array to detect copy number changes,
genome-wide, at unprecedented resolution. Currently, this type of analysis will only detect
gains and losses of chromosomal material and will not detect balanced rearrangements,
such as translocations and inversions which are hallmark aberrations seen in many types of
leukemia and lymphoma.
Spectral karyotyping

Spectral karyotyping is an image of colored chromosomes. Spectral karyotyping involves

FISH using multiple forms of many types of probes with the result to see each
chromosome labelled through its metaphase stage. This type of karyotyping is used
specifically when seeking out chromosome arrangement.
FISH has a large number of applications in molecular biology and
medical science, including gene mapping, diagnosis of chromosomal abnormalities, and
studies of cellular structure and function. Chromosomes in three-dimensionally preserved
nuclei can be "painted" using FISH. In clinical research, FISH can be used for prenatal
diagnosis of inherited chromosomal aberrations, postnatal diagnosis of carriers of
genetic disease, diagnosis of infectious disease, viral and bacterial
disease, tumor cytogenetic diagnosis, and detection of aberrant gene expression. In
laboratory research, FISH can be used for mapping chromosomal genes, to study
the evolution of genomes (Zoo FISH), analyzing nuclear organization, visualization of
chromosomal territories and chromatin in interphase cells, to analyze dynamic nuclear
processes, somatic hybrid cells, replication, chromosome sorting, and to study tumor
biology. It can also be used in developmental biology to study the temporal expression of
genes during differentiation and development. Recently, high resolution FISH has become
a popular method for ordering genes or DNA markers within chromosomal regions of
interest.
 PRINCIPLE
A fluorescent-labelled specific rRNA targeted oligonucleotide probe is
used for the direct identification, independent of culture, and quantification of the
microorganism ( Amman et al., 1995). The use of in situ hybridization for counting
and identifying organisms was proposed by Olsen et al. (1986) twenty years ago.
The first assays were performed using radioactively labelled oligonucleotides
(Giovannoni et al., 1988), and later by using fluorescent probes, which yield superb
spatial resolution and can be detected very simply by using epifluorescent
microscopy and fluorescent in situ hybridization (FISH) (Amann et al., 1990).
Microorganisms are made permeable to oligonucleotide probes by
fixation with aldehydes (formalin, paraformaldehyde, glutaraldehyde) or alcohols
(methanol, ethanol) (Amann et al., 1995).
When a fluorescence probe (labelled with a fluorescence dye, e.g.
Cy3 or fluorescein) is placed in contact with permeable cells in fixed physico-
chemical conditions, the fluorescent probe hybridizes with the specific intracellular
target site in the ribosomes (Amann et al., 1995).
Later, a universal fluorochrome that binds DNA, such as DAPI, is
used to evaluate the total number of viable cells in the sample. With
epifluorescence microscopy the hybridized cells can be observed and counted, and
after changing the filter, the total number of viable cells is counted.
 How does fish work

Put the chromosomes on a microscope slide and denature them.

Denature the probe and add it to the microscope slide, allowing the probe hybridize to
its complementary site.
Wash off the excess probe and observe the chromosomes under a fluorescent
microscope
.
The probe will show as one or more fluorescent signals in the microscope, depending
on how many sites it can hybridize to.
 How many types of probes for FISH
Generally, researchers use three different types of FISH probes, each of which has a
different application:

Locus specific probes bind to a particular region of a chromosome. This type of

probe is useful when researchers have isolated a small portion of a gene and want to
determine on which chromosome the gene is located.

Alphoid or centromeric repeat probes are generated from repetitive

sequences found in the middle of each chromosome. Researchers use these probes to
determine whether an individual has the correct number of chromosomes. These probes
can also be used in combination with "locus specific probes" to determine whether an
individual is missing genetic material from a particular chromosome.

Whole chromosome probes are actually collections of smaller probes, each of

which binds to a different sequence along the length of a given chromosome. Using
multiple probes labelled with a mixture of different fluorescent dyes, scientists are able to
label each chromosome in its own unique colour. The resulting full-colour map of the
chromosome is known as a spectral karyotype. Whole chromosome probes are
particularly useful for examining chromosomal abnormalities, for example, when a piece
of one chromosome is attached to the end of another chromosome.
 procedure

Warm to 37c ,vortex centrifuge for 1to3 sec

Denature probe for 10 min at65c,and hold at 37c for 30-60 min
Prepare new slides with fresh metaphasespreads,which have beeb fixed with 3:1
methanol: acetic acid
Dehydrate by serial ethanol washing for 2min eachin 70%ethanol ,70% 90% 90%and 5
min00%.age for 60 min at 65c
Denature slide by incubatingin pre-warmed denaturation solution at 65c for2min
Quench slides in ice cold 70%ethanol for 4min and dehydrate by serial ethanol washing
for 2min eachin 70% ethnaol ,70% 90% 90% and 5 min 100%.dry it at room temperature
Apply probe onto the slide ,apply coverslips and remove air bubbles by gently pushing on
cover slip with pencil. seal it with rubber cement
Place the slide in an air tight,prewarmed humidified chamber and incubate at night in
the dark at 37c
 ADVANTAGES
oRapid technique and large number of cells can be scored in short period
oEfficiency of hybridisation and detection is high
oSensitivity and specifity is high
oCytogenetic data can be obtained from non dividing or terminally differentiated cells
oCytogeneic data can be obtained from poorsamplesthat contain too few cells for routine
cytogenic analysis
oMethods has ben adapted for automated systems

DISADVANTAGES
oRestricted to those abnormalities hat can be detected with currently avliabke probe
oOnly one or a few abnormalities can be assessed simultaneously
oDue to failure to detect signal FISH is higher sensetive for trisomy but less sensitive for
detecting chromosome loss or deletion
oRequires flourescence microscopy and an image analysis system
 Scanning electron microscope
A scanning electron microscope (SEM) is a type of electron microscope that produces
images of a sample by scanning the surface with a focused beam of electrons. The
electrons interact with atoms in the sample, producing various signals that contain
information about the sample's surface topography and composition. The electron beam is
scanned in a raster scan pattern, and the beam's position is combined with the detected
signal to produce an image. SEM can achieve resolution better than 1 nanometer.
Specimens can be observed in high vacuum in conventional SEM, or in low vacuum or wet
conditions in variable pressure or environmental SEM, and at a wide range of cryogenic or
elevated temperatures with specialized instruments.
The most common SEM mode is detection of secondary electrons emitted by atoms
excited by the electron beam. The number of secondary electrons that can be detected
depends, among other things, on specimen topography. By scanning the sample and
collecting the secondary electrons that are emitted using a special detector, an image
displaying the topography of the surface is created
 History
An account of the early history of SEM has been presented by McMullan. Although Max
Knoll produced a photo with a 50 mm object-field-width showing channeling contrast by
the use of an electron beam scanner, it was Manfred von Ardenne who in 1937 invented a
true microscope with high magnification by scanning a very small raster with a demagnified
and finely focused electron beam.
Ardenne applied the scanning principle not only to achieve magnification but also to
purposefully eliminate the chromatic aberration otherwise inherent in the electron
microscope.
He further discussed the various detection modes, possibilities and theory of SEM, together
with the construction of the first high magnification SEM.
Further work was reported by Zworykin's group, followed by the Cambridge groups in the
1950s and early 1960s headed by Charles Oatley, all of which finally led to the marketing of
the first commercial instrument by Cambridge Scientific Instrument Company as the
"Stereoscan" in 1965, which was delivered to DuPont.
Principles and capacities
The signals used by a scanning electron microscope to produce an image result from
interactions of the electron beam with atoms at various depths within the sample.
Various types of signals are produced including secondary electrons(SE), reflected
or back-scattered electrons (BSE), characteristic X-rays and light
(cathodoluminescence) (CL), absorbed current (specimen current) and transmitted
electrons. Secondary electron detectors are standard equipment in all SEMs, but it is
rare that a single machine would have detectors for all other possible signals.
In secondary electron imaging, or SEI, the secondary electrons are emitted from very
close to the specimen surface. Consequently, SEM can produce very high-resolution
images of a sample surface, revealing details less than 1 nm in size. Back-scattered
electrons (BSE) are beam electrons that are reflected from the sample by elastic
scattering. They emerge from deeper locations within the specimen and consequently
the resolution of BSE images is less than SE images. However, BSE are often used in
analytical SEM along with the spectra made from the characteristic X-rays, because the
intensity of the BSE signal is strongly related to the atomic number (Z) of the specimen.
BSE images can provide information about the distribution of different elements in the
sample. For the same reason, BSE imaging can image colloidal gold immuno-labels of 5
or 10 nm diameter, which would otherwise be difficult or impossible to detect in
secondary electron images in biological specimens.
 Sample preparation

A spider sputter-coated in gold, having been prepared for viewing with an SEM.
Low-voltage micrograph (300 V) of distribution of adhesive droplets on a Post-it note. No
conductive coating was applied: such a coating would alter this fragile specimen.
Samples for SEM have to be prepared to withstand the vacuum conditions and high energy
beam of electrons, and have to be of a size that will fit on the specimen stage. Samples are
generally mounted rigidly to a specimen holder or stub using a conductive adhesive. SEM is
used extensively for defect analysis' of semiconductor wafers, and manufacturers make
instruments that can examine any part of a 300 mm semiconductor wafer. Many
instruments have chambers that can tilt an object of that size to 45° and provide
continuous 360° rotation.
Nonconductive specimens collect charge when scanned by the electron beam, and especially
in secondary electron imaging mode, this causes scanning faults and other image artifacts

For conventional imaging in the SEM, specimens must be electrically conductive, at least at the
surface, and electrically grounded to prevent the accumulation of electrostatic charge. Metal
objects require little special preparation for SEM except for cleaning and conductively
mounting to a specimen
Nonconducting specimens may be imaged without coating using an environmental SEM
(ESEM) or low-voltage mode of SEM operation. In ESEM instruments the specimen is
placed in a relatively high-pressure chamber and the electron optical column is
differentially pumped to keep vacuum adequately low at the electron gun. The high-
pressure region around the sample in the ESEM neutralizes charge and provides an
amplification of the secondary electron signal.] Low-voltage SEM is typically conducted in
an FEG-SEM because field emission guns(FEG) are capable of producing high primary
electron brightness and small spot size even at low accelerating potentials. To prevent
charging of non-conductive specimens, operating conditions must be adjusted such that
the incoming beam current is equal to sum of out coming secondary and backscattered
electrons currents a condition that is more often met at accelerating voltages of 0.3–4 kV.
Synthetic replicas can be made to avoid the use of original samples when they are not
suitable or available for SEM examination due to methodological obstacles or legal issues.
This technique is achieved in two steps: (1) a mold of the original surface is made using a
silicone-based dental elastomer, and (2) a replica of the original surface is obtained by
pouring a synthetic resin into the mold.
Embedding in a resin with further polishing to a mirror-like finish can be used for both
biological and materials specimens when imaging in backscattered electrons or when
doing quantitative X-ray microanalysis.
The main preparation techniques are not required in the environmental SEM outlined
below, but some biological specimens can benefit from fixation.
 Biological samples
• For SEM, a specimen is normally required to be completely dry, since the
specimen chamber is at high vacuum. Hard, dry materials such as wood, bone,
feathers, dried insects, or shells (including egg shells[) can be examined with little
further treatment but living cells and tissues and whole, soft-bodied organisms
require chemical fixation to preserve and stabilize their structure.
• Fixation is usually performed by incubation in a solution of a buffered chemical
fixative, such as glutaraldehyde, sometimes in combination
with formaldehyde and other fixatives,[23and optionally followed by postfixation
with osmium tetroxide. The fixed tissue is then dehydrated. Because air-drying
causes collapse and shrinkage, this is commonly achieved by replacement
of water in the cells with organic solvents such as ethanol or acetone, and
replacement of these solvents in turn with a transitional fluid such as
liquid carbon dioxide by critical point drying. The carbon dioxide is finally removed
while in a supercritical state, so that no gas–liquid interface is present within the
sample during drying.
• The dry specimen is usually mounted on a specimen stub using an adhesive such
as epoxy resin or electrically conductive double-sided adhesive tape, and sputter-
coated with gold or gold/palladium alloy before examination in the microscope.
Samples may be sectioned (with a microtome) if information about the organism's
internal ultra structure is to be exposed for imaging.
• If the SEM is equipped with a cold stage for cryo microscopy, cryofixation may be
used and low-temperature scanning electron microscopy performed on the
cryogenically fixed specimens.[ Cryo-fixed specimens may be cryo-fractured under
vacuum in a special apparatus to reveal internal structure, sputter-coated and
transferred onto the SEM cryo-stage while still frozen.
 SEM Properties
The Scanning Electron Microscope developed by professor Dr. Charles Oatlev with
the assistance of graduate students in the 1950s, are one of the three types of
electron microscopes (EM).
Electron microscopes utilize the same basic principles as light microscopes, but
focus beams of energetic electrons rather than photons, to magnify an object.
SEMs consist of the following components:
Electron Source
Thermionic Gun
Field Emission Gun
Electromagnetic and/or Electrostatic Lenses
Vacuum chamber
Sample chamber and stage
Computer
Detectors (one or more)
Secondary Electron Detector (SED)
Backscatter Detector
Diffracted Backscatter Detector (EBSD)
X-ray Detector (EDS)
In addition, SEMs require a stable power supply, vacuum and cooling system,
vibration-free space and need to be housed in an area that isolates the instrument
from ambient magnetic and electric fields.

 SEM Imaging
A Scanning Electron Microscope provides details surface information by tracing a sample in
a raster pattern with an electron beam.
The process begins with an electron gun generating a beam of energetic electrons down
the column and onto a series of electromagnetic lenses.
These lenses are tubes, wrapped in coil and referred to as solenoids.
The coils are adjusted to focus the incident electron beam onto the sample; these
adjustments cause fluctuations in the voltage, increasing/decreasing the speed in which
the electrons come in contact with the specimen surface.
Controlled via computer, the SEM operator can adjust the beam to control magnification as
well as determine the surface area to be scanned.
The beam is focused onto the stage, where a solid sample is placed. Most samples require
some preparation before being placed in the vacuum chamber.
Of the variety of different preparation processes, the two most commonly used prior to
SEM analysis are sputter coating for non-conductive samples and dehydration of most
biological specimens.
In addition, all samples need to be able to handle the low pressure inside the vacuum
chamber.
 SEM Applications
SEMs have a variety of applications in a number of scientific and industry-related
fields, especially where characterizations of solid materials is beneficial.

In addition to topographical, morphological and compositional information, a Scanning

Electron Microscope can detect and analyze surface fractures, provide information in
microstructures, examine surface contaminations, reveal spatial variations in chemical
compositions, provide qualitative chemical analyses and identify crystalline structures.
SEMs can be as essential research tool in fields such as life science, biology, gemology,
medical and forensic science, metallurgy.
In addition, SEMs have practical industrial and technological applications such as
semiconductor inspection, production e of miniscule products and assembly of
microchips for computers.

SEM Advantages
Advantages of a Scanning Electron Microscope include its wide-array of applications,
the detailed three-dimensional and topographical imaging and the versatile
information garnered from different detectors.
SEMs are also easy to operate with the proper training and advances in computer
technology and associated software make operation user-friendly.
This instrument works fast, often completing SEI, BSE and EDS analyses in less than five
minutes. In addition, the technological advances in modern SEMs allow for the generation
of data in digital form.
Although all samples must be prepared before placed in the vacuum chamber, most SEM
samples require minimal preparation actions.

SEM Disadvantages
The disadvantages of a Scanning Electron Microscope start with the size and cost.
SEMs are expensive, large and must be housed in an area free of any possible electric,
magnetic or vibration interference.
Maintenance involves keeping a steady voltage, currents to electromagnetic coils and
circulation of cool water.
Special training is required to operate an SEM as well as prepare samples.
The preparation of samples can result in artifacts. The negative impact can be
minimized with knowledgeable experience researchers being able to identify artifacts
from actual data as well as preparation skill .
 Transmission electron microscope
A TEM image of a cluster of poliovirus. The polio virus is 30 nm in diameter

Operating principle of a Transmission Electron

Microscope

Transmission electron microscopy (TEM, also sometimes conventional transmission

electron microscopy or CTEM) is a microscopy technique in which a beam of electrons is
transmitted through a specimen to form an image. The specimen is most often an ultrathin
section less than 100 nm thick or a suspension on a grid. An image is formed from the
interaction of the electrons with the sample as the beam is transmitted through the
specimen. The image is then magnified and focused onto an imaging device, such as
a fluorescent screen, a layer of photographic film, or a sensor such as a charge-coupled
device.
Transmission electron microscopes are capable of imaging at a significantly
higher resolution than light microscopes, owing to the smaller de Broglie wavelength of
electrons. This enables the instrument to capture fine detail—even as small as a single
column of atoms, which is thousands of times smaller than a resolvable object seen in a
light microscope. Transmission electron microscopy is a major analytical method in the
physical, chemical and biological sciences. TEMs find application in cancer
research, virology, and materials science as well
as pollution, nanotechnology and semiconductor research.
At lower magnifications TEM image contrast is due to differential absorption of electrons
by the material due to differences in composition or thickness of the material.
Sample preparation

Sample preparation in TEM can be a complex procedure.TEM specimens should be less

than 100 nanometers arable to the mean free path of the electrons that travel through
the samples, which may be only a few tens of nanometers. Preparation of TEM
specimens is specific to the material under analysis and the type of information to be
obtained from the specimen
Unlike neutron or X-Ray radiation the electrons in the beam interact readily with the
sample, an effect that increases roughly with atomic number squared (z2). High quality
samples will have a thickness that is compA sample of cells (black) stained with osmium
tetroxide and uranyl acetate embedded in epoxy resin (amber) ready for sectioning.
Materials that have dimensions small enough to be electron transparent, such as
powdered substances, small organisms, viruses, or nanotubes, can be quickly prepared
by the deposition of a dilute sample containing the specimen onto films on support grids.
Biological specimens may be embedded in resin to withstand the high vacuum in the
sample chamber and to enable cutting tissue into electron transparent thin sections. The
biological sample can be stained using either a negative staining material such as uranyl
acetate for bacteria and viruses, or, in the case of embedded sections, the specimen may
be stained with heavy metals, including osmium tetroxide. Alternately samples may be
held at liquid nitrogen temperatures after embedding in vitreous ice.
• Materials science a common use is for examining the fresh fracture surface of metal alloys.
• Modifications
• The capabilities of the TEM can be further extended by additional stages and detectors,
sometimes incorporated on the same microscope.
• Scanning TEM
• A TEM can be modified into a scanning transmission electron microscope (STEM) by the
addition of a system that rasters the beam across the sample to form the image, combined
with suitable detectors. Scanning coils are used to deflect the beam, such as by an
electrostatic shift of the beam, where the beam is then collected using a current detector
such as a Faraday cup, which acts as a direct electron counter. By correlating the electron
count to the position of the scanning beam (known as the "probe"), the transmitted
component of the beam may be measured. The non-transmitted components may be
obtained either by beam tilting or by the use of annular dark field detectors.
• Low-voltage electron microscope.
• A low-voltage electron microscope (LVEM) is operated at relatively low electron accelerating
voltage between 5–25 kV. Some of these can be a combination of SEM, TEM and STEM in a
single compact instrument. Low voltage increases image contrast which is especially
important for biological specimens. This increase in contrast significantly reduces, or even
eliminates the need to stain. Resolutions of a few nm are possible in TEM, SEM and STEM
modes. The low energy of the electron beam means that permanent magnets can be used
as lenses and thus a miniature column that does not require cooling can be used.
 TEM History
Ernst Ruska developed the first electron microscope, a TEM, with the assistance of Max
Knolls in 1931. After significant improvements to the quality of magnification, Ruska joined
the Sieman’s Company in the late 1930s as an electrical engineer, where he assisted in the
manufacturing of his TEM.
TEMs consist of the following components:
An electron source
Thermionic Gun
Electron beam
Electromagnetic lenses
Vacuum chamber
2 Condensers
Sample stage
Phosphor or fluorescent screen
Computer
A Transmission Electron Microscope functions under the same basic principles as an optical
microscope.
In a TEM, electrons replace photons, electromagnetic lenses replace glass lenses and images
are viewed on a screen rather than through an eyepiece.
 TEM Imaging
A Transmission Electron Microscope produces a high-resolution, black and white image from
the interaction that takes place between prepared samples and energetic electrons in the
vacuum chamber.
Air needs to be pumped out of the vacuum chamber, creating a space where electrons are able
to move.
The electrons then pass through multiple electromagnetic lenses. These solenoids are tubes
with coil wrapped around them.
The beam passes through the solenoids, down the column, makes contact with the screen
where the electrons are converted to light and form an image.
The image can be manipulated by adjusting the voltage of the gun to accelerate or decrease
the speed of electrons as well as changing the electromagnetic wavelength via the solenoids.
The coils focus images onto a screen or photographic plate.
During transmission, the speed of electrons directly correlates to electron wavelength; the
faster electrons move, the shorter wavelength and the greater the quality and detail of the
image.
The lighter areas of the image represent the places where a greater number of electrons were
able to pass through the sample and the darker areas reflect the dense areas of the object.
These differences provide information on the structure, texture, shape and size of the sample.
To obtain a TEM analysis, samples need to have certain properties. They need to be sliced thin
enough for electrons to pass through, a property known as electron transparency.
Samples need to be able to withstand the vacuum chamber and often require special
 TEM Applications
A Transmission Electron Microscope is ideal for a number of different fields such as life sciences,
nanotechnology, medical, biological and material research, forensic analysis, gemology and
metallurgy as well as industry and education
TEMs provide topographical, morphological, compositional and crystalline information.
The images allow researchers to view samples on a molecular level, making it possible to
analyze structure and texture
This information is useful in the study of crystals and metals, but also has industrial applications.
TEMs can be used in semiconductor analysis and production and the manufacturing of
computer and silicon chips.
Technology companies use TEMs to identify flaws, fractures and damages to micro-sized objects;
this data can help fix problems and/or help to make a more durable, efficient product.
Colleges and universities can utilize TEMs for research and studies.
Although electron microscopes require specialized training, students can assist professors and
learn TEM techniques.
Students will have the opportunity to observe a nano-sized world in incredible depth and detail.
 ADVANTAGES
A Transmission Electron Microscope is an impressive instrument with a number of
advantages such as:
TEMs offer the most powerful magnification, potentially over one million times or more.
TEMs have a wide-range of applications and can be utilized in a variety of different
scientific, educational and industrial fields.
TEMs are able to yield information of surface features, shape, size and structure
They are easy to operate with proper training.

DISADVANTAGES
Some cons of electron microscopes include:
TEMs are large and very expensive.
Laborious sample preparation.
Potential artifacts from sample preparation.
Operation and analysis requires special training.
Samples are limited to those that are electron transparent, able to tolerate the vacuum
chamber and small enough to fit in the chamber.
TEMs require special housing and maintenance.
Images are black and white.
G-banding, G banding, or Giemsa banding is a technique used in cytogenetics to produce a
visible karyotype by staining condensed chromosomes. It is useful for identifying genetic
diseases through the photographic representation of the entire chromosome
complement. The metaphasechromosomes are treated with trypsin(to partially digest the
chromosome) and stained with Giemsa stain. Heterochromatic regions, which tend to be
rich with adenine and thymine (AT-rich) DNA and relatively gene-poor, stain more darkly in
G-banding. In contrast, less condensed chromatin—which tends to be rich
with guanine and cytosine (GC-rich) and more transcriptionally active—incorporates less
Giemsa stain, and these regions appear as light bands in G-banding. The pattern of bands
are numbered on each arm of the chromosome from the centromere to the telomere. This
numbering system allows any band on the chromosome to be identified and described
precisely. The reverse of G-bands is obtained in R-banding. Banding can be used to identify
chromosomal abnormalities, such as translocations, because there is a unique pattern of
light and dark bands for each chromosome.
It is difficult to identify and group chromosomes based on simple staining because the
uniform colour of the structures makes it difficult to differentiate between the different
chromosomes. Therefore, techniques like G-banding were developed that made "bands"
appear on the chromosomes. These bands were the same in appearance on
the homologous chromosomes, thus, identification became easier and more accurate. The
less condensed the chromosomes are, the more bands that appear when G-banding. This
means that the different chromosomes are more distinct in prophase than they are in
metaphase acid-saline-Giemsa protocol reveals G-banding.
Chromosome Banding: Technique # 2. G-Bands:
The technique of G-banding involves Giemsa staining following pre-treatment with weak
trypsin solution, urea or protease. It provides greater detail than C-banding. It was first used
for human chromosomes by Summer et al. in 1971. G-bands may reflect a stronger chromatin
condensation. However, this technique is not suitable for plant chromosomes.
Chromosome Banding: Technique # 3. Q-Bands:
The method of Q-banding was developed by Caspersson et al. in 1968. The chromosomes
stained with Quinacrine mustard show bright and dark zones under UV light. This technique is
used to identify human and mice chromosomes.
Chromosome Banding: Technique # 4. N-Bands:
The technique of N-banding was originally described by Matsui and Sasaki in 1973. Briefly, air-
dried chromosomes slides are stained for 90 minutes with Giemsa (diluted 1 : 10 in 1/15 M
phosphate buffer at pH 7.0) following extraction with 5% trichloroacetic acid at 95°C for 30
minutes and then 0.1 NHCl at 60″C for 30 minutes.
The N-bands are generally located at the secondary constriction, satellites, centromeres,
telomeres and heterochromatic segments. It is suggested that the N-bands represent certain
structural non-histone proteins specifically linked to the nucleolar organizer region of the
eukaryotic chromosomes.
The N- banding patterns have been used for the location of nucleolar regions in the different
organisms, such as, mammals, birds, amphibians, fishes, insects and plants. N-banding
patterns differ in the chromosomes of different species.
In 1980, Islam used this method to identify the barley chromosomes from those of wheat in
the reciprocal wheat-barely F, hybrids, and to detect translocations between the wheat and
barley chromosomes. He also used this technique to isolate lines possessing a pair of barely
chromosomes substituted for particular pair of wheat chromosomes.
A modified Giemsa-N-banding technique was developed by Singh and Tsuchiya in 1982 for
the identification of barley chromosomes. This method is a combination of acetocarmine
staining and Giemsa-N-banding. After processing according to this method, the centromeric
region looks like a “diamond-shaped” structure; this is not seen in other techniques.
Early metaphase or prometaphase chromosomes are more suitable for this staining as they
show better banding pattern than the chromosomes at mid-metaphase in somatic cells.
Chromosome Banding: Technique # 5. Other Techniques of
Chromosome Banding:
Besides the above, there are other techniques for chromosome banding, e.g., R-banding
(Reverse Giemsa banding). H-banding, and T-banding (Terminal banding). Chromosome
banding patterns can be used not only for the identification of individual chromosomes of an
organism but also to establish evolutionary relationships between different species.
Banding patterns in human, chimpanzee, gorilla and orangutan have indicated that the
evolutionary relationship between human and chimpanzee is closer than that between
human and gorilla. It has further indicated that humans have a more distant evolutionary
relationship with orangutans.
 Banding type Staining method

C-banding constitutive heterochromatin

G-banding Giemsa stain

Q-banding quinacrine

R-banding reverse Giemsa staining

T-banding telomeric
patterns lend each chromosome a distinctive appearance so the 22 pairs of human nonsex
chromosomes and the X and Y chromosomes can be identified and distinguished without
ambigchromosome banding
Chromosome banding: The treatment of chromosomes to reveal characteristic patterns of
horizontal bands like bar codes.
The banding uity. Banding also permits the recognition of chromosome deletions (lost
segments), chromosome duplications (surplus segments) and other types of structural
rearrangements of chromosomes.
The distinctive pattern, visible in a light microscope, that results from the selective binding of
certain dyes to individual chromosomes.
Banding patterns are patterns of light and dark transverse bands on chromosomes. The light
and dark bands become apparent by staining the chromosome with a chemical solution and
then viewed under a microscope. These bands describe the location of genes on a
chromosome.
Chromosomes in metaphase can be identified using certain staining techniques, so called
banding. Cells are cultured and then stopped in metaphase to maximize the number of suitable
cells. They are then spread on a slide, stained with a suitable dye and visualized in the
microscope. Most conventional cytogenetic analyses depend on the karyotyping of banded
metaphase chromosomes.
The banding techniques fall into two principal groups: 1) those resulting in bands distributed
along the length of the whole chromosome, such as G-, Q- and R-bands and 2) those that stain
a restricted number of specific bands or structures. These latter include methods which
reveal centromeric bands, C-bands, and nucleolus organizer regions, NOR's (at terminal
regions of acrocentric chromosomes). C-banding methods do not permit identification of every
chromosome in the somatic cell complement, but can be used to identify specific
chromosomes.
G- and R- bands can be bright field or fluorescent.
Bright field G-bands
These G-bands are most commonly used. They take their name from the Giemsa
dye, but can be produced with other dyes. In G-bands, the dark regions tend to
be heterochromatic, late-replicating and AT rich. The bright regions tend to
be euchromatic, early-replicating and GC rich.
Bright field R-bands
These R-bands are approximately the reverse of G-bands (the R stands for "reverse").
The dark regions are euchromatic and the bright regions are heterochromatic.

Fluorescent G- and R-bands

These bands are the photographic negative of the bright field versions. i.e. the
reverse of the bright field G-bands and R-bands.

Q-bands are like fluorescent G-bands, but certain heterochromatic regions are
more brightly stained with Q-banding.
 CONCLUSION
After completing this project I gained a lot of knowledge on
the topics sem tem chromosome painting and banding.
This project gave me a lot of knowledge on terms I have never
heard. I came to know about its advantages disadvantages procedure and my more.
I hope this project will be liked and all mistakes will be acknowledged.
 REFRENCES

•Environmental Scanning Electron Microscopy (VP-ESEM). John Wiley & Sons.

• McMullan, D. (2006). "Scanning electron microscopy 1928–1965“.
•McMullan, D. (1988). "Von Ardenne and the scanning electron microscope".
•Wikipedia