This document discusses fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH), which are molecular cytogenetic techniques used to localize DNA sequences on chromosomes. FISH uses fluorescent probes to detect specific DNA or RNA sequences on chromosomes. GISH uses total genomic DNA as a probe to detect specific chromosomes. Both techniques overcome limitations of conventional cytogenetics and have various applications, including gene mapping, analyzing structural abnormalities, and detecting aneuploidy. The document discusses the principles, methods, advantages and limitations of FISH and GISH.
DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCINGPuneet Kulyana
This presentation will give you a brief idea about the various DNA sequencing methods and various strategies used for genome sequencing and much more vital information related to gene expression and analysis
Introduction
History
Genetic mapping
DNA Markers
Physical mapping
Importance
Drawback
Conclusion
References
uses genetic techniques to construct maps showing the positions of genes and other sequence features on a genome.
Genetic techniques include cross-breeding experiments or, in the case of humans, the examination of family histories (pedigrees).
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to investigate the presence of small, submicroscopic chromosomal changes that are beyond the resolution of karyotype analysis.
This PowerPoint presentation explain the concept,process and application of Fluorescence insitu hybridization.
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.
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCINGPuneet Kulyana
This presentation will give you a brief idea about the various DNA sequencing methods and various strategies used for genome sequencing and much more vital information related to gene expression and analysis
Introduction
History
Genetic mapping
DNA Markers
Physical mapping
Importance
Drawback
Conclusion
References
uses genetic techniques to construct maps showing the positions of genes and other sequence features on a genome.
Genetic techniques include cross-breeding experiments or, in the case of humans, the examination of family histories (pedigrees).
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to investigate the presence of small, submicroscopic chromosomal changes that are beyond the resolution of karyotype analysis.
This PowerPoint presentation explain the concept,process and application of Fluorescence insitu hybridization.
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.
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
Modern cytogenetic tools in crop improvementShreyas A
it includes FISH, GISH and their recent modifications such as comparative genome hybridization, chromosome painting, spectral karyotyping, multicolour FISH, fiber FISH and Q-FISH
INTRODUCTION
Hybridization stages
probe synthesis
Probe marking
Target DNA processing
Target DNA denaturation
Target DNA transfer to solid carrier
Visualization
CONCLUSIONS
REFERENCES
MOLECULAR BIOLOGY TECHNIQUES USED IN ZOONOTIC DISEASE Nataraju S M
Zoonotic pathogens cause diseases and death both in human & animals which ultimately leads to man power and economic loss of the country. Traditional diagnostic methods identify a pathogen based on its phenotype.
The correct assessment of a clinical isolate takes more time. Faster and simpler methods of diagnosis is of great advantage. That is why molecular biology technique is the first and foremost choice .
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
What are molecular probes ? What is RNA probe? Applications of Probe ?
Presented By: M.Tech Biotechnology Students (IIT Guwahati). Bharat Bhushan Negi
Manoj Kumar S.
Umesh Kushwah
Medisetti Rajmohan Naidu
Vikkurthi Rajesh
An honest effort to present molecular marker in easiest way both informative and conceptual. Hybridization based (non-PCR) and PCR based markers are discussed to the point with suitable diagram.
Taxonomy is the branch of science concerned with the classification of organisms. A taxonomic designation is more than just a name. Ideally, it reflects evolutionary history and the relationship between organisms. Traditionally, taxonomic classification has relied upon morphological features and physiological characteristics. However, for bacterial taxonomy, phenotypic approaches have proven insufficient. Unrelated bacteria can exhibit identical traits, closely related bacteria can have divergent features, and methods for accurate identification may be too cumbersome for routine use. In contrast, molecular taxonomy approaches use data derived from hereditary material and provide a robust view of genetic relatedness. Advances in technology have been accompanied by improvements in the cost, speed, and availability of molecular methods. Here, we provide a brief history of approaches to prokaryotic classification and describe how molecular taxonomy is redefining our understanding of bacterial evolution and the tree of life.
Quantitative Trait LOci (QTLs) Mapping: Basics procedure, principle and MethodsMahesh Hampannavar
Basics procedure, principle, and Methods of QTL mapping, preparation of linkage mapping, calculation of recombination frequency and LOD value.
For more information on Calculation of LOD value and single marker analysis contact me personally on following mail id: mahi5295@gmail.com
Introduction
Homeologous gene- in polyploidy
Repeatable patterns - after allopolyploidization
Common mechanisms in the creation of variation
Problems associated - polyploid crop improvement.
Strategies for overcoming problems
its cover some points regarding Mendel Experiment and detail molecular characterization of seven characters of garden pea. Its journey from 1866 to 2016 or factors to gene or mendel to molecular and its 150 year long journey
it cover almost all content in cis/intragesis, right from introduction definition, explanation, production of marker free transgenic, intragenic vector construction, regulatory guide lines, current and future status, limitation, advantage over existing technique, swot analysis etc
its very useful for your seminar and presentations. it contain lot of picture, table, figure for your easy understanding
thank you
Mahesh
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Toxic effects of heavy metals : Lead and Arsenicsanjana502982
Heavy metals are naturally occuring metallic chemical elements that have relatively high density, and are toxic at even low concentrations. All toxic metals are termed as heavy metals irrespective of their atomic mass and density, eg. arsenic, lead, mercury, cadmium, thallium, chromium, etc.
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
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Exposé invité Journées Nationales du GDR GPL 2024
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
1. FISH and GISH: molecular cytogenetic tools
and their applications
MAHESH R HAMPANNAVAR
PhD scholar
Dept of Genetics and Plant
breeding
1 Mahesh R Hampannavar (mahi5295gmail.com)
2. Disadvantages of conventional
cytogenetic technique
2
Always study at particular stage of cell division
Most of aberration are observed during meiosis cell division
With Q, C, G, N and R bands are restricted to centromere or telomere or
heterochromatine regions.
Mahesh R Hampannavar (mahi5295gmail.com)
3. Disadvantages of molecular techniques
Normal hybridization requires the isolation of DNA or RNA
PCR
separating it on a gel
blotting it into nitrocellulose.
Ex situ methods
3 Mahesh R Hampannavar (mahi5295gmail.com)
4. In situ hybridization:
In situ Hybridization(ISH) is a powerful method to localize nucleic
acid sequences in vivo i.e. in tissues, cells, organelles, nuclei or
chromosomes by using appropriate probes.
With ISH, nucleic acids are localized in their original or proper
place.
4 Mahesh R Hampannavar (mahi5295gmail.com)
5. Different types localized by ISH
DNA sequence
• Repetitive seq.
• Unique seq
• Whole
chromosome
or part of
chromosome
• Whole genome
RNA seq.
• Helps to study
the spatial and
temporal
pattern of
genes
expression
Viral sequence
• Forms the
basis of
diagnosis of
several viral
diseases.
5 Mahesh R Hampannavar (mahi5295gmail.com)
6. Principles underlying in situ reactions:
Most of in situ reactions have a 1o and 2o step.
Reaction is carried out on chromosomes in situ involve a
1o reaction and that defines the specificity of the reaction,
20 reaction that provides the means for detecting the product of
reaction . Chemicals used in preparation of chromosomes can
determine the specificity of a primary reaction.
6 Mahesh R Hampannavar (mahi5295gmail.com)
7. FISH and GISH Techniques
modification of in situ hybridization technique
A fluorescent molecule is deposited at the site of in situ
hybridization location of genes or DNA can be visualized on
chromosomes:- Fluorescence in situ hybridization (FISH)
Total genomic DNA is used as probe in hybridization
experiments :- Genomic in situ hybridization (GISH)
7 Mahesh R Hampannavar (mahi5295gmail.com)
8. Fluorescence in situ Hybridization (FISH)
A process which distinctly paints and detects RNA as well as DNA
Structures, numbers and location in place in the cell or in situ.
FISH may be used with:
Morphologically preserved chromosome preparations (Metaphase).
Fixed cells or tissue sections (Interphase)
Aids in gene mapping, toxicological studies, analysis of
chromosome structural aberrations, and ploidy determination
8 Mahesh R Hampannavar (mahi5295gmail.com)
9. Advantages of Interphase FISH
Interphase cells for FISH do not require culturing of the cells and
stimulating division to get metaphase spreads
interphase FISH is faster than methods using metaphase cells
valuable for analysis of cells that do not divide well in culture, including fixed
cells.
200–500 cells can be analyzed microscopically using FISH
the sensitivity of detection is higher than that of metaphase procedures, which
commonly examine 20 spreads.
9 Mahesh R Hampannavar (mahi5295gmail.com)
10. Metaphase FISH
Uses fluorescent probes that bind to metaphase
chromosomal regions or to whole chromosomes.
Whole chromosome paints: Probes that cover the
entire chromosome, are valuable for detecting small
rearrangements that are not apparent by regular
chromosome banding.
Telomeric and centromeric probes are also applied to
metaphase chromosomes to detect aneuploidy and
structural abnormalities
10 Mahesh R Hampannavar (mahi5295gmail.com)
11. Genomic in situ hybridization (GISH)
Genomic in situ hybridization (GISH) Is a cytogenetic technique
that allows the detection and localization of specific nucleic acid
sequences on morphologically preserved chromosomes using
genomic DNA of donor specie as probe.
An unlabeled DNA of parental specie is used as competitor DNA .
GISH for plants…was developed in 1987 by M.D. Bennett and
J.S. Heslophorizon
11 Mahesh R Hampannavar (mahi5295gmail.com)
12. Importance of FISH and GISH
detect specific nucleotide sequences within cells and tissues
unique link among the studies of cell biology, cytogenetics, and
molecular genetics.
it is possible to detect single-copy sequences on chromosome with
probes shorter than 0.8 kb.
12 Mahesh R Hampannavar (mahi5295gmail.com)
13. FISH essentially involves seven steps:-
Probe DNA-Characterization.
Nick translation labeling of probe DNA.
Purification of labeled DNA probe.
Chromosome preparation.
In situ hybridization.
Detection of hybridization.
Microphotography.
13 Mahesh R Hampannavar (mahi5295gmail.com)
14. GISH essentially involve eight steps: -
Probe DNA
isolation and shearing of probe DNA
Isolation and sizing the competitor DNA
Nick translation labeling of probe DNA
Purification of labeled DNA probe
Chromosome preparation
In situ hybridization
Detection of hybridization
Microphotography.
DNA from a test organism that
is denatured and then used in
vitro hybridization experiment
s in which it competes
with DNA (homologous) from
a reference organism; used
to determine the relationship o
f the test organism to
the reference organism.
14 Mahesh R Hampannavar (mahi5295gmail.com)
15. Steps involved in FISH and GISH
Denature the chromosomes
Denature the probe
Hybridization
Fluorescence staining
Examine slides or store in the dark
15 Mahesh R Hampannavar (mahi5295gmail.com)
17. ON THE DENATURATION OF CHROMOSOMAL DNA
IN SITU
Denaturation of chromosomal DNA for fluorescence in situ
hybridization (FISH) is an essential step
shorter denaturation time in 70% formamide/2 × SSC at 720C
provides sufficient denaturation, where the hydrogen bonds are
broken between the purines and pyrimidines of the double helix.
denaturation in alkali (0.07 N NaOH at room temperature) and
formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C
denaturation in HCl (0.24 M) at room temperature and 60%
formamide: 2 × 10−4 M EDTA (pH 8) at 55 °C. The presence of 4%
formaldehyde in the denaturation buffer prevents DNA loss.
Barbera et al.,
Ramesh et al., 2009
17 Mahesh R Hampannavar (mahi5295gmail.com)
18. Probes
complimentary sequences of
nucleotide bases
20-40 base pairs or be up to
1000 bp
Small probe- penetration
RNase resistant
Types of probe that can be
used in performing in situ
hybridization
Oligonucleotide probes
Single stranded DNA probes.
Double stranded DNA probes
18 Mahesh R Hampannavar (mahi5295gmail.com)
19. Labels…
Radioactive labels Non-radioactive labels
Radioactive labels are the
isotopes which emit β-
particles and are detected by
autoradiography . E.g. 35S ,
32P , 3H
Non-radioactive labelling
procedures are of two types:-
Direct ISH Indirect ISH
Labeling of
probes
Non
radioactive
labeling
Direct
method
Indirect
method
Radioactive
labeling
19 Mahesh R Hampannavar (mahi5295gmail.com)
20. Radioactive probe
•Traditionally oligonucleotide probes have been radiolabeled.
•Radiolabeled probes are still the choice for many workers.
•Radiolabeled probes are visualized by exposure of the tissue section or cells against
photographic film which is then developed.
•When using radiolabeling, waste disposal and containment measures must be given
thought and it must be remembered that the useful shelf life of labeled probe is
inherently dependent on the half life of the radionucleotide.
20 Mahesh R Hampannavar (mahi5295gmail.com)
21. Non-radioactive probe
Direct probe Indirect probe
label is incorporated directly
into nucleic acid probe so that
hybridization site could be
visualized immediately after
hybridization .
the label in the probe cannot be
detected immediately after
hybridization .
second molecule called reporter is
required to detect the label in
probe
This reporter molecule is
conjugated with signal
generating system which makes
the visualization of probe possible .
Can be: - one step or two step (with
2o reporter )
21 Mahesh R Hampannavar (mahi5295gmail.com)
22. Non radioactive probe
Chemical labelling
Acetylaminofluorine
Mercury
Enzymatic labelling
• Biotin
• Digoxigenin
22 Mahesh R Hampannavar (mahi5295gmail.com)
23. Reporter Molecules in non-radioactive probe
Biotin is detected by avidin or streptavidin
Digoxigenin is detected by anti-digoxigenin antibodies.
AAF is detected by anti-acetylaminofluorene antibodies.
Mercury is detected by ligands having an immunogenic group
which can bind to same antibody
23 Mahesh R Hampannavar (mahi5295gmail.com)
24. Fluorochromes
• They get excited by
light of one
wavelength and
emit light of another
wavelength
• which is observed as
fluorescence of
different colors.
Enzymes
• Enzymes work by
catalyzing the
precipitation of a
visible product at
hybridization site.
Metals
• Colloidal gold which
is conjugated to
antibodies.
• Can be visualized
with both light and
electron
microscope
Signal Generating System:
24 Mahesh R Hampannavar (mahi5295gmail.com)
25. Fluorochromes
Fluorochrome Excite Fluorescene
Fluorescene isothiocyanate (FITC) Blue Green
Tetramethyl rhodamine isothiocyanate (TRITC) Green Red
Texas red or sulphorhodamine Green Deep red
Amino methyl coumarine acetic acid (AMCA) UV Blue
25 Mahesh R Hampannavar (mahi5295gmail.com)
26. Enzyme Substrate Color of ppt
Horseradish peroxidase Diamino benzidine(DAB) Red
Alkaline phosphatase
5-Bromo 4-chloro 3-indolyl
phosphate (BCIP)
Blue
Enzymes.
26 Mahesh R Hampannavar (mahi5295gmail.com)
27. Example- Detection by Biotin
• Biotin, is first introduced enzymatically
into NA probe.
• Probe hybridized to target NA.
• Then avidin, conjugated to same signal
generating system, (say FITC) is
introduced.
• Detected by green colored fluorescene
of FITC.
• To enhance the signal strength avidin
can further be detected by biotin-
antiavidin conjugate.
• Then again avidin, conjugated to some
signal generating system is introduced.
27 Mahesh R Hampannavar (mahi5295gmail.com)
28. •non-radioactive labels used successfully with in situ hybridization
include
•Biotin, digoxin and digoxigenin (DIG), alkaline phosphatase
and the fluorescent labels, fluorescein (FITC), Texas Red and
rhodamine.
•Since these nonradioactive labels have no inherent "decay"
kinetics, -20C for later use for as long as 1-2 years with careful
storage.
28 Mahesh R Hampannavar (mahi5295gmail.com)
29. Hybridization issues
In situ hybridization presents a unique set of problems
as the sequence to be detected will be at a lower concentration,
be masked because of associated protein,
protected within a cell or cellular structure.
Therefore, in order to probe the tissue or cells of interest one has to increase the
permeability of the cell and the visibility of the nucleotide sequence to the
probe without destroying the structural integrity of the cell or tissue.
29 Mahesh R Hampannavar (mahi5295gmail.com)
30. Permeablization
Three common reagents used to permeabilize tissue are
HCl,
detergents (Triton or SDS)
Proteinase K.
30 Mahesh R Hampannavar (mahi5295gmail.com)
32. The factors that influence the hybridization of the oligonucleotide
probe to the target mRNA are:
Temperature
pH
monovalent cation concentration
presence of organic solvents
33 Mahesh R Hampannavar (mahi5295gmail.com)
33. The following is a typical hybridization solution with a hybridization
temperature of around 370C
Dextran sulphate - reduces the amount of hydrating water for dissolving the
nucleotides and therefore effectively increases the probe concentration in
solution resulting in higher hybridization rates.
Formamide and DTT (dithiothreitol) - These are organic solvents which
reduce the thermal stability of the bonds allowing hybridization to be
carried out at a lower temperature.
34 Mahesh R Hampannavar (mahi5295gmail.com)
34. SSC (NaCl + Sodium citrate) - Monovalent cations interact mainly with the
phosphate groups of the nucleic acids decreasing the electrostatic interactions
between the two strands.
EDTA - This is a chelator and removes free divalent cations from the
hybridization solution, because they strongly stabilize duplex DNA.
35 Mahesh R Hampannavar (mahi5295gmail.com)
36. Technical developments
Although the principle steps of the FISH procedure have
stayed the same
little technical modification adopted in plant cytogenetics
Tyramid-FISH
Three dimensional FISH
FISH on super- stretched chromosome
FISH on DNA fibers
37 Mahesh R Hampannavar (mahi5295gmail.com)
37. Tyr FISH
The sensitivity of fluorescence in situ hybridization (FISH) for
mapping plant chromosomes of single copy DNA sequences is
limited.
ultra-sensitive FISH with tyramide signal amplication
Increase the detection sensitivity of FISH experiment
Use of signal amplification molecule i.e. peroxidase
conjugated antibody
Use this as first layer of signal detection
38 Mahesh R Hampannavar (mahi5295gmail.com)
38. Then to use fluorochrome- labeled tyramides as peroxidase substrates
to generate and deposit many fluorochromes close to the in situ
bound peroxidase.
Sensitivity increased 10-100 times
DNA probes smaller than 1 kb were successfully visualized
Chromosome 1 of transgenic shallot line 7-1 L4, accession number 54 probed
with 13.3 kb pTOK233 plasmid DNA. (a) Conventional FISH; (b) Tyr-FISH.
DNA was counterstained with DAPI.
39 Mahesh R Hampannavar (mahi5295gmail.com)
39. Frequency of single-copy T-DNA detection on metaphase chromosomes of transgenic shallots (Allium
cepa) using Tyr-FISH and conventional FISH
40 Mahesh R Hampannavar (mahi5295gmail.com)
40. Three dimensional
FISH
Bass et al., 1997 in maize
Precise position of DNA probe on chromosome within the nuclie.
It doesn’t helps in mapping technique
It helps to identify the spitial position of probe during cell division
Stacks the FISH signals are taken and composed into 3 dimensional image
41 Mahesh R Hampannavar (mahi5295gmail.com)
42. FISH on super- stretched
chromosome
The ability to isolate single chromosomes, which represent small units of nuclear genome,
is priceless in many areas of plant research including cytogenetics, genomics, and
proteomics.
Flow cytometry is the only technique which can provide large quantities of pure
chromosome fractions suitable for downstream applications
physical mapping, preparation of chromosome-specific BAC libraries, sequencing,
and optical mapping.
43 Mahesh R Hampannavar (mahi5295gmail.com)
43. Flow cytometry as an analytical and
preparative technique
Flow cytometry, accompanied by sorting, permits isolation of individual
chromosomes for further study, and generates highly pure and chromosome-
specific DNA preparations.
FCM involves the passage of chromosome suspensions through the focus of
intense light sources, typically lasers, using one or two DNA-specific
fluorochromes to provide fluorescent signals related to the DNA content and
base-pair composition of the individual chromosomes.
The end products are “flow karyotypes” providing one- or two-dimensional
representations of the distributions of the different chromosomes (histograms or
44 Mahesh R Hampannavar (mahi5295gmail.com)
44. FISHIS of pasta wheat cv Creso chromosome suspensions.
a) Chromosome suspensions hybridized with (GAA)7-FITC;
b) flow-sorted chromosomes 3B, 4B and 5B following
hybridization with (AG)12-Cy3
45 Mahesh R Hampannavar (mahi5295gmail.com)
45. Representative images of chromosomes flow-sorted from three HOSUT
(Transgenic lines of hexaploid wheat carrying barley (Hordeum vulgare)
sucrose transporter HvSUT1 (SUT) gene) lines
46 Mahesh R Hampannavar (mahi5295gmail.com)
46. Figure 2. Biparametric dot plot analysis of pasta wheat cv Creso chromosomes.
Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, et al. (2013) FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome
Flow Sorting Made Easy. PLOS ONE 8(2): e57994. https://doi.org/10.1371/journal.pone.0057994
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057994
47 Mahesh R Hampannavar (mahi5295gmail.com)
47. Flow sorted plant chromosome can be stretched
to more than 100 times their original size
It facilitate the brighter signal in FISH
Mapping resolution of up to 70kb
48 Mahesh R Hampannavar (mahi5295gmail.com)
48. FISH on DNA fiber
Due to higher molecular weight or large insert size of DNA clones
(2.5-3.5kb/um)
Fiber DNA method provide fine mapping resolution of up to a
few kilobases.
So this method used in various mapping
This method used to measure the size of physical gaps in
mapping
49 Mahesh R Hampannavar (mahi5295gmail.com)
50. Limits of Fiber FISH
Not applicable to large genome size crops like Wheat, because
extensive amount of repetitive DNA
Problem to distinguish short fiber FISH (<1kb) from background
stain
51 Mahesh R Hampannavar (mahi5295gmail.com)
52. Chromosome mapping
Species specific, repetitive sequence, ribosomal sequence and unique sequence.
Ribosomal genes have the great value in karyotyping and comparative genomics
5S and 18S-5.8S-26S rDNA multi gene family
53 Mahesh R Hampannavar (mahi5295gmail.com)
53. Case study 1
The 5S ribosomal RNA genes were mapped to mitotic
chromosomes of Arabidopsis thaliana by fluorescence in
situ hybridization (FISH).
ecotypes, Columbia and Wassilewskija
54 Mahesh R Hampannavar (mahi5295gmail.com)
56. Genome analyze
Hybrid plants, allopollyploidy and recombinant inbred line
mFISH and whole total genomic DNA probe is promising approach
in amphidiploid
Helps to know the progenitors of allopolyploidy
57 Mahesh R Hampannavar (mahi5295gmail.com)
57. Common wheat, Triticum aestivum, is an allohexaploid species consisting of
three different genomes (A, B, and D).
Double ditelo 1A line of cv. Chinese Spring wheat
Biotinylated total genomic DNA of the diploid A genome progenitor Triticum
urartu, digoxigenin-labeled total genomic DNA of the diploid D genome
progenitor Aegilops squarrosa, and nonlabeled total genomic DNA of one of
the possible B genome progenitors Ae. speltoides were hybridized in situ to
metaphase chromosome spreads of Triticum aestivum cv. Chinese Spring.
58 Mahesh R Hampannavar (mahi5295gmail.com)
58. The hybridization sites of the A genome probe were detected by yellow fluorescence, while those of
the D genome probe were detected by orange fluorescence. The B genome chromosomes were
neither labeled yellow nor orange but appeared faint brown as a result of cross-hybridization of
the A and B genome probes59 Mahesh R Hampannavar (mahi5295gmail.com)
59. Multicolour genomic in situ hybridization was carried out in wheat*rye
hybrids and in a wheat*rye translocation line.
Different hybridization conditions and mixture compositions were used,
and A, B and D genomes of hexaploid wheat as well as the R genome of
rye were distinguished simultaneously in somatic cells.
60 Mahesh R Hampannavar (mahi5295gmail.com)
61. Phylogenetic relationship
GISH offers new opportunities in phylogenetic and taxonomic study
The 5S rRNA genes of higher plants are organized into clusters of tandem
repeats with thousands of copies at one or more position in the genome.
Each repeat consist of a highly conserved in a species
The variation in sizes and sequences of the NTS of the 5S rRNA gene was
found to be useful for phylogenetic reconstruction of spices.
62 Mahesh R Hampannavar (mahi5295gmail.com)
62. 5S rRNA gene probe was obtained using
total genomic DNA of Allium stulosum.
63 Mahesh R Hampannavar (mahi5295gmail.com)
64. Analysis of Somaclonal variations:
In tissue culture
Chromosomal breakage and DNA transposition leads to change in
karyotype.
Examination of the chromosomal distribution of 5S and 18S-26S
rRNA is useful to identifying the types of genomic changes
65 Mahesh R Hampannavar (mahi5295gmail.com)
65. The specific aims of the present
investigation were:
(1) to generate plants from callus
derived from flower bud explants
of A. tuberosum
(2) to analyze the karyotypic
change of At30 in comparison with
the wild type by using 5S and 18S-
5.8S-26S rDNA as probes.
66 Mahesh R Hampannavar (mahi5295gmail.com)
66. Among these variants, At30 revealed the highest percentage of
aneuploid cells (26.4%).
One of the most interesting findings was the fact that At30 showed
better viability and growth than wild-type plant and other
aneuploid plants during the culture period for two generations.
At30 was phenotypically normal, developed extensive roots and
grew into healthy plants with normal flowering
67 Mahesh R Hampannavar (mahi5295gmail.com)
67. FISH patterns using the 5S and 18S-5.8S-26S rDNA probes of wild A. tuberosum metaphase chromosome (A and
B) and At30 (C and D) (bar=10µm). Digoxigenin-labelled 5S rDNA probe detected with antidigoxigenin-
rhodamine conjugate (red) (A and C) and biotin-labelled 18S-5.8S-26S rDNA probe detected with avidin-FITC
conjugate (green) (B and D). Numbers indicate corresponding chromosomes showing rDNA hybridization sites.
to analyze the karyotypic change of At30 in
comparison with the wild type by using 5S and
18S- 5.8S-26S rDNA as probes.
68 Mahesh R Hampannavar (mahi5295gmail.com)
68. Interspecific and intergenic crosses
Alien chromosome, chromosome segment and gene can be identify
and characterization by FISH and GISH.
Detection of chromosomal aberration
Almost aberration observed during meiosis
Than plant its more important to human being
mFISH for all 24 chromosome by using the human genomics library
69 Mahesh R Hampannavar (mahi5295gmail.com)
70. Wheat-rye hybrids were produced by crossing
winter wheat (Triticum aestivum L.) cultivar
“Xiaoyan No.6”and winter rye (Secale cereale
L.) cultivar “German White”.
“Xiaoyan No.6 × rye” were detected by GISH.
Eight lines were found carrying rye
chromosomes or s egments
Two highly repeated DNA sequences
pSc119.2 and pAs1 were labeled with
Fluorogreen and Fluororedvia nick
translation respectively.
Two-color fluorescent in situ hybridization
(FISH) was used to identify the genome
composition and individual chromosome of the
hybrids using pSc119.2 labled with fluorogreen
common wheat Thinopyrum ponticum
(2n=70)
“Xiaoyan
No.6”
“German
White”.
F1
doubling
Hybrid detected by
GISH
three
addition lines
one substitution
line
four
translocation
71 Mahesh R Hampannavar (mahi5295gmail.com)
72. Chromosome specific painting
Determination karyotype based on chromosome size, centric index
and banding pattern has its own limitation.
Discrimination of similar chromosomes.
Helps to discriminate the same size chromosome within its own
genome
73 Mahesh R Hampannavar (mahi5295gmail.com)
73. Four Vicia species (V. sativa, V.
grandiflora, V. pannonica and V.
narbonensis).
DNA sequences (18S–25S and 5S
rDNA, telomeres) and
genus‐specific satellite repeats
(VicTR‐A and VicTR‐B)
the VicTR‐A and ‐B sequences, in
particular, produced highly
informative banding patterns that
alone were sufficient for
discrimination of all chromosomes.
74 Mahesh R Hampannavar (mahi5295gmail.com)
75. Idiograms of Vicia sativa (A), V.
grandiflora (B), V. pannonica
(C) and V. narbonensis (D).
Presence and distribution of
18S–25S rRNA genes (orange),
5S rRNA genes (yellow),
VicTR‐A (red), VicTR‐B
(green) and telomeres (white) is
indicated.
76 Mahesh R Hampannavar (mahi5295gmail.com)
76. Schematic comparison of
chromosome sizes among Vicia
sativa (A), V.
grandiflora (B), V. pannonica (C)
and V. narbonensis (D). Relative
chromosome lengths were derived
from the data in Table 2 and scaled
according to the nuclear DNA
content of the species (Table 1).
77 Mahesh R Hampannavar (mahi5295gmail.com)
77. Spectral karyotyping (SKY) and multiple
fluorescent hybridization (M-FISH)
By mixing combinations of five fluors and using special
imaging software, can distinguish all 23 chromosomes by
chromosome specific colors.
This type of analysis can be used to detect abnormalities
that affect multiple chromosomes as is sometimes found in
cancer cells or immortalized cell lines.
78 Mahesh R Hampannavar (mahi5295gmail.com)
78. limitations of FISH
The inability to identify chromosomal changes other than those at the specific
binding region of the probe.
Preparation of the sample is critical in interphase FISH analysis
to permeabilize the cells for optimal probe target interaction
to maintain cell morphology.
Cannot detect small mutations.
Probes are not yet commercially available for all chromosomal regions
79 Mahesh R Hampannavar (mahi5295gmail.com)
79. Relativelly expensive
Multi color FISH can only be used successfully on polyploid with at least one
known proginator species
Closely related genomes in certain allopolyploids cannot be discriminate using
GISH technique
mFISH is less sensitive and shows a lower degree of detection resolution than
single color FISH due to multiple exposure photographs
80 Mahesh R Hampannavar (mahi5295gmail.com)
The fluorescence intensity emissions from chromosomes stained with DAPI (DNA content) and labeled by FISHIS with GAA-FITC are joint together into a bi-parametric dot plot where each dot represents a single particle (blue: DNA stained by DAPI; green: (GAA)7-FITC labeling). Similar particles with a similar fluorescence emission are clustered and can then be enclosed into a sorting region for flow sorting and single-type chromosome isolation (colored regions). Panels showing the chromosome content from each relevant dot plot sorting region display various purity levels. The sorting purity is presented as a percentage of the main sorted chromosome in respect to the total number of the sorted population. Chromosome region distribution is directly proportional to the whole intensity of the fluorescence hybridization pattern. Different colored regions R1–R5 were used to assess the MESF (Molecules of Equivalent Soluble Fluorescein) values (Figure S5). The (GAA)7 oligonucleotides hybridize less with the A-genome chromosomes than the B-genome ones. As expected, the A-genome chromosomes are found within regions R1–R3 and the B-genome chromosomes are enclosed in regions R4 and R5.
Mitotic metaphase chromosomes from root meristems of A. thaliana ecotypes Ler (a) and Col (b), stained with propidium iodide, showing FITC
signals after in situ hybridization with biotinylated 5S rDNA as a probe.
Both comprise pseudo-color images by confocal laser microscopy (upper) and drawings derived from them with chromosome numbers (lower). Bar - 2 pm
Identification of A. thaliana chromosomes.
(a) DAPI-stained metaphase chromosomes of Let, showing green fluorescein signals from the 5S rDNA and red Cy-3 signals from the 18S-5.8S-25S rDNA. Chromosomes were identified by the FISH pattern and numbered according to the linkage group. Bar = 2 pm. (b) A karyotype showing the locations of the 5S rDNA, 18S-5.8S-25S rDNA, centromeric 180 bp repeats (CEN), and seven cosmid clones: g6838 (chromosome 1, map (mp) 109.4); g6842 (chromosome 2, mp 58.5); g3838 (chromosome 3, mp 9.9); g2440 (chromosome 3, mp 39.3); g4539 (chromosome 4, mp 49.8); g6843 (chromosome 5, mp 32.5); g4028 (chromosome 5, mp 57.7).
establish fine physical maps of Arabidopsis
Fluorescence micrographs of wheat–rye hybrids (a–c) and the translocation line M233 (d). (a) Mitotic metaphase with GISH identification of A genome (yellow–brown), B genome (brown), D genome (red) and R genome (yellow–green). (b) Somatic interphase in which genome identification is indicated. (c) Mitotic metaphase with GISH identification of A genome (red), B genome (yellow), D genome (brown) and R genome (green); arrows indicate bright scattered signals observed on wheat genomes. (d) Somatic metaphase with simultaneous GISH identification of A genome (green), B genome (brown), D genome (red) and R genome (bright green) and pTa71 probe (red) hybridization; asterisks indicate the 1BL/1RS translocated chromosomes. In (a), (c) and (d), arrowheads indicate the 4AL/7BS translocation.
Localization of repetitive sequences on metaphase chromosomes of Vicia sativa (A, B), V. grandiflora (C–E), V. pannonica (F–H) and V. narbonensis (I–L).
5S rDNA (A) and VicTR‐B (B) sequences on V. sativa chromosomes.
VicTR‐B (red) and 18S–25S rDNA (green) (C), 5S rDNA (D), and telomeric probes (E) on V. grandiflora. 18S–25S rDNA (F), VicTR‐A (red) and 5S rDNA (green) (G), and telomeric sequences (H) on V. pannonica. VicTR‐B (red) and 18S–25S rDNA (green) (I), VicTR‐B (red) and 5S rDNA (green) (J), VicTR‐A (red) and VicTR‐B (green) (K), and telomeric repeats (L) on V. narbonensis chromosomes. Sequences were localized using FISH except for VicTR repeats in B, C and G, which were visualized using PRINS. Chromosomes were counterstained with DAPI (blue). Bar = 10 µm.