GENOMIC AND FLUROSCENCE IN-SITU HYBRIDIZATION AND ITS IMPORTANCE

1. SANDESH G M
PALB 8079
I Ph.D, GPB
GKVK

2. Chromosome
 Chromosomes are rod-shaped, filamentous bodies.
 Present in the nucleus and become visible during cell division.
 They are the carriers of genes or unit of heredity.
 Chromosomes are not visible in active nucleus due to their high water content,
but are clearly seen during cell division.

4.  In the past decade the development of genetic and physical maps has greatly benefited
from improvements in molecular biological tools.
 Cytogenetic or chromosome maps serve as intermediates between physical and
genetic maps in displaying the approximate positions of genes and molecular markers,
relative to structural markers, such as centromeres, telomeres, heterochromatin bands
and secondary constrictions.
 Most of the individual chromosomes generally lack unique banding patterns, and so
cannot be identified.
 In addition, spontaneous structural and numerical chromosome mutants are scarce,
and are laborious and time consuming to generate.
 An alternative analysis strategy is in situ hybridization (ISH), which allows distinct
positioning of unique sequences and repetitive DNA on chromosomes.

5. CYTOGENETIC
TECHNIQUES
Conventional techniques (chromosome
banding techniques)
 Q-banding (Caspersson)
 G-banding (trypsin and Giemsa)
 C-banding (Pardue and Gall-1970)
 R-banding
 NOR-banding (Goodpasture)
 T-banding
Molecular techniques
(in situ Hybridisation techniques)
 Fluorescent in situ Hybridisation
(FISH)
 Genomic in situ Hybridisation
(GISH)
 Spectral Karyotyping (SKY)
 Comparative Genomic Hybridisation
(CGH)
 Chromosome painting

6.  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 on
chromosome will be identified.

7.  The preparation of biological material that has to be investigated.
 Probes are labeled.
 Both probes target nucleic acid are denatured.
 Single stranded probe gets hybridized to the region where it found sequences
complementary to it.
 Hybridization is detected
 Hybridization is visualized.

8. DNA sequences RNA sequences Viral sequences
Repetitive seq.
Unique seq.
Whole chromosome or
a part of chromosome
Whole genome
Helps to study the spatial
& temporal patters of
gene expression.
Forms the basis of
diagnosis of several viral
diseases

9.  Probes are fragments of DNA that were isolated, purified, amplified and labeled with
fluorophore, with sites for interaction with antibody or avidin.
 Probes can vary in length from a few base pairs for synthetic oligonucleotides to larger
than one Mbp.
 Probe size is important because longer probes hybridize more specifically than shorter
ones.
 There are 3 main types of probes for FISH:
1) locus specific probes,
2) centrometric repeat probes -- repeated DNA sequences,
3) whole chromosome probes
 Fluorescent dyes- Cy 5 (far red), Cy 3 (orange), FLUOS (green).

10. Centromeric
(satellite) probes
Locus specific probes
Whole chromosome painting
probes

11. Nucleic acid type: Probe synthesis Complexities of
probe sequences:
DNA
RNA
Cloned
Synthetic
PCR
For repeated seq.
For single copy
For whole chromosome
For part of chromosome
For total genomic DNA

12. Chemical labelingEnzymatic labeling
 Acetylaminofluorine
 Mercury
 Biotin
 Digoxigenin
Radioactive labels
Radioactive labels are the isotopes
which emit β- particles and are detected
by autoradiography .
E.g: 35S , 32P , 3H
Non-radioactive labels
Non-radioactive labeling
procedures are of two types:-
 Direct ISH
 Indirect ISH

13. Direct ISH
 Label is incorporated directly into
nucleic acid probe so that
hybridization site could be
visualized immediately after
hybridization .
Indirect ISH
 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.

14. 1. Fluorochromes:
They get excited by light of one wavelength and emit light of another
wavelength which is observed as fluorescence of different colors.
Fluorochrome Fluorescene
Fluorescene isothiocyanate (FITC) Green
Tetramethyl rhodamine isothiocyanate
(TRITC)
Red
Texas red or sulphorhodamine Deep red
Amino methyl coumarine acetic acid
(AMCA)
Blue

15. 2. Enzymes :
Enzymes work by catalyzing the precipitation of a visible product at
hybridization site.
3. Metals :
Colloidal gold which is conjugated to antibodies. Can be visualized with
both light and electron microscope
Enzyme Substrate Color
Horseradish peroxidase Diamino benzidine(DAB) Red
Alkaline phosphatase 5-Bromo 4-chloro 3-indolyl
phosphate (BCIP)
Blue

16. In multiple labeling more than one probe can be employed simultaneously on target
nucleic acid.
1. Sequential multiple labeling :
To determine the relationship of different sequences with respect to each other.
2. Simultaneous multiple labeling (Indirect method) :
To identify different chromosomes simultaneously.
3. Simultaneous multiple labeling (Direct method) :
To identify different genomes simultaneously.

17. 17
 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.
+
+
ssProbe with biotin
Avidin with SGS as FITC
ssDNA

18. FISH
 Fluorescence in situ hybridization (FISH) :
A fluorescent molecule is deposited at the site of in situ hybridization
location of genes or DNA can be visualized on chromosomes.
 Genomic in situ hybridization (GISH) :
Total genomic DNA is used as probe in hybridization experiments

19. FLUROSCENCE IN SITU
HYBRIDIZATION
(FISH)
 Is a cytogenetic technique that allows detection
and localization of specific nucleic acid
sequences on morphologically preserved
chromosomes.
 It uses florescent probes that bind only to those
parts of chromosomes which show a high
degree of sequence similarity.
 Aids in gene mapping, toxicological studies,
analysis of chromosome structural aberrations,
and ploidy determination.

20. HEATING
DENATURATION
PROBE WITH
FLUROSCENT LABEL
COOLING
HYBRIDISATION
NUCELIC ACID

21. • Probe DNA-Characterization.
• Nick translation labeling of
probe DNA.
• Purification of labeled DNA
probe.
• Chromosome preparation.
• In situ hybridization.
• Detection of hybridization.
• Microphotography.

22. Can visualize specific cytogenetic abnormalities (copy number aberrations)
 chromosomal deletion, amplification, translocation
Each fluorescently labeled probe that hybridizes to a cell nucleus in the tissue of interest will
appear as a distinct fluorescent dot
 Diploid nuclei will have two dots
 If there is duplication in the region of interest, the gain will result in more than two dots.
 If there is a loss in the region of interest, one or zero dot will result.
 Was often used during Metaphase but is now used on Interphase chromosomes as well.
Advantage :
 Less labor-intensive method for confirming the presence of a DNA segment within an entire
genome than other conventional methods like Southern blotting

23.  Interphase cells for FISH do not require culturing of the cells and stimulating
division to get metaphase threads.
 Interphase FISH is faster than methods using metaphase cells
 Valuable for analysis of cells that do not divide well in culture, including fixing 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.

24.  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:
Todetect aneuploidy and structural abnormalities

25. Advantages:
 The highest possible resolution.
 conspicuous differentiation of euchromatin, chromomeres and larger heterochromatin
blocks.
 Typical pairing configurations, such as loops, multivalents and trisomics are helpful in
providing information about specific chromosomal regions.
Disadvantages :
 Raising a plant to flowering stage requires more time and effort.
 selection of anthers with PMCs at right stage makes the production of pachytene-spreads
relatively time consuming and laborious.
 In polyploids difficult - complex configurations, asynapsis and chromosome stickiness.
 tracing and identification of individual chromosomes in pachytene nuclei . Eg; Allium ,
Lilium and rye.

26.  Genomic in situ hybridization (GISH), which is a modification of fluorescent in situ
hybridization.
 GISH for plants was developed in 1987 by M.D. Bennett and J.S. Heslop-harrison
 It 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.
 GISH is a technique that allows distinguishing the genomes in a cell.
 An unlabeled DNA of parental specie is used as competitor DNA.

27.  It is possible to differentiate the genomes in a hybrid.
 To the study of hybrid lineages detecting the amount of introgressed chromatin
during the production of new lineages.
 Evolution of polyploids.
 The GISH has direct applications on the understanding of the genome evolution of
partial allopolyploids and recombinant inbred lines.
 GISH can be applied to the analysis of the meiotic behavior in hybrids and
polyploids, providing information concerning the relationship between species.
 GISH is used for the purpose of distinguishing chromosomes from different genitors
or from different genomes in interspecific/intergeneric hybrids or allopolyploids

28.  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 experiments in which it
competes with DNA (homologous)
from a reference organism; used to
determine the relationship of the test
organism to the reference organism.

29. • DNA of a genitor involved in the formation of a hybrid is used at the same time as an unlabeled DNA
from another genitor, at a higher concentration, which serves as a blocking DNA, hybridizing with the
sequences in common with both genomes.
• Total genomic DNA must be broken into smaller fragments, which can be done by using restriction
enzymes, autoclaving, or sonicating.
• The use of blocking DNA is usually necessary in hybrids derived from close species, due to the high
degree of homology shared between the species that originated the hybrid, which may enable the
probe to label the genomes of both genitors indistinctly
• Lycopersicon esculentum and L. peruvianum – More blocking DNA. (Parokonny et al., 1997
• Dendranthema nankingense and Tanacetum vulgare – No Blocking DNA. (Tang et al., 2011)
• By adjusting the blocking DNA concentration for each species, in accordance with the degree of
genetic proximity, it is possible to assure that the probe stains only one of the genomes.

30. A. Direct and indirect probe labeling.
B. Fragmentation of the blocking DNA.
C. Slide preparation.
D. Probe and blocking DNA denaturation
in a hybridization mixture.
E. Addition of the hybridization mixture
with the probe and the blocking DNA.
F. Denaturation of the chromosome DNA.
G. In situ hybridization of probe and
blocking DNA in the target sequence of the
chromosome.
H. Detection of the probe in the
chromosome DNA of one parent, in an
indirect labeling.
I. Chromosome DNA molecule of the sec‐
ond parent associated to the unlabeled
blocking DNA.
J. Visualization of hybridization signals
associated to a probe (green) in a
fluorescence microscope. Unmarked
chromosomes are visualized with a
counter-staining (blue).

31.  Telomeres form the ends of eukaryotic chromosomes and serve as protective caps that
keep chromosomes structure independency and completeness.
 The first plant telomere DNA was isolated from Arabidopsis thaliana and was shown
to have tandemly repeated sequence 5´-TTTAGGG-3 .
 Objective :
To identify the telomere and measure its length in cotton.

32.  Materials :
Results :
 Telomeric repeats of cotton cross-hybridize with that of Arabidopsis.
 The terminal restriction fragment (TRF) showed that the telomere length of cultivated
cotton was close to 20 kb and was larger than those of wild cotton species whose
telomere length ranged from 6 to 20 kb.

33.  Signal intensity varied between arms of the
same chromosome, between chromosomes
of the same complement (B and E).
 The chromosomes of G. herbaceum also
showed weak signal intensity (A).
 Most chromosomes of the two tetraploid
cotton species showed a strong signal
intensity with the telomere probe (F and G).
 In seven diploid cotton species, the
chromosomes of G. somalense and G.
longicalyx showed relatively strong signal
intensity with the telomere probe (C and D).
 The telomere signal at the ends of
chromosomes in G. nelsonii and
G. raimondii was the weakest and amplified
by by cy3 and biotin-cy3 two times to make
the signal detectable (H and I).
FISH of cotton metaphase chromosomes with telomere probes
G.
herbaceum
G.
anomalum
G.
somalense
G. longicalyx G. bickii TM-1
XinHai 7 G. nelsonii G. raimondii

34.  It is well known that the intensity of in situ hybridization signal corresponds with the
homology or length of target DNA sequence.
 Four kinds of restrict enzyme Hae III, Sau3A I, Hha I, and Hap II, which recognize sites
only four bases, were used in TRF to digest subtelomere completely.
 Three cultivated cotton G. herbaceum, TM-1, and Xinhai 7 showed the telomere length of
close to 20 kb.
 G. anomalum and G. somalin have relatively long telomere repeats between 10 and 20 kb.
 G. raimondii and G. longicalyx showed repeats of intermediate length close to 10 kb.
 G. nelsonii and G. bickii showed short repeats.
 The telomere length may vary not only among cotton species but also between each
chromosome in the same species. The strong hybridization signal in tetraploid cotton may
indicated that the telomere DNA length of tetraploid cotton was longer than that of diploid
cotton.

35. OBJECTIVE :
 FISH-To find out the accurate localisation of repetitive DNA sequences at chromosomal sub-
arm level in Brassica species.
 GISH-Identification of alien chromatin in intergenomic rapeseed hybrids and characterization of
parental genome components in rapeseed hybrids.

36. Materials :
Here they described the application of methods for GISH analysis of intergeneric
Brassica hybrids for the characterization of backcross progeny from B. napus hybrids
exhibiting nematode resistance introduced from Raphanus sativus and Phoma
resistance from Sinapis arvensis (charlock mustard) and Coincya monensis (Wild
mustard) respectively.

37.  Mitotic metaphases were generated from seedlings of diverse Brassica cultivars for FISH
and from hybrid plants for GISH. Cytological preparations were made from young root
tips.
 For multicolour FISH, a 25S rDNA clone from Arabidopsis thaliana and the 5S rDNA
subunit from Beta vulgaris were labelled by nick translation with the fluorochromes Cy3
and fluorescein, respectively.
 For GISH probes, genomic DNA extracted from Raphanus sativus, Sinapis arvensis and
Coincya monensis was directly labelled with Cy3 and mixed with a 50-fold volume of
unlabelled, sheared B. napus genomic DNA .
 Composite fluorescence images were obtained using a fluorescence microscope fitted with
specific single-band filters for DAPI, FITC and Cy3.

38.  Multicolour FISH was used for
the co-localisation of 25S and 5S
rDNA loci on mitotic metaphase
and prometaphase chromosomes
from various Brassica species (a).
 In B. napus, colocalisation of the
six 25S rDNA loci with the seven
5S rDNA loci (b) enables the
reliable identification of 10
chromosome pairs(c).

39.  Identification with GISH of addition
chromosomes in BC3 individuals
from B. napus crosses with
Raphanus sativus, Sinapis arvensis
and Coincya monensis, respectively.
 In both the R. sativus and S. arvensis
crosses, fertile BC3 individuals
exhibiting the desired resistance
character were found which
contained monosomic addition
chromosomes from the respective
donor genome.

40.  In several plant groups, especially those with polyploid complexes as Triticum (the wheat
genus, Poaceae), related species can be used as important sources of genes.
 In the tribe Triticeae as a whole, which comprises other important cereals as barley
(Hordeum vulgare) and rye (Secale cereale), there are high rates of successful interspecific
hybridization .
 Phenotypic constitution and characterization of chromosome types among accessions of a
germplasm collection.

41. Materials :
T. Aestivum x S. cereale,
2n = 6x = 42 2n = 2x = 14,
AABBDD RR
Triticale
2n = 6x = 42, AABBRR
 Genomic DNA from rye was used as probe and wheat genomic DNA was used as
blocking agent in a proportion of 1:10 (probe:blocking agent).

42. Metaphase cell of triticale (2n = 56) is shown with the 14 chromosomes from rye detected by GISH.
This cell was hybridized with blocking DNA of wheat and rye DNA probe labeled with digoxigenin
and detected with FITC (fluorescein isothiocyanate).
A. The chromosomes are counterstained with DAPI.
B. Shows the image capture of the same chromosomes with the fluorescence filter for FITC.
C. Shows the superposition of both images.

43.  Objective:
To develop a GISH procedure for identifying chromosomes or chromosome segments of wild species
in the background of cultivated sunflower
 Materials:
1.BC4F1 progeny with 2n=35 of H. californicus (2n=102) × HA 410 (2n=34.
2. BC4F1,2n=35 and BC4F2, 2n=34 progeny from cytoplasmic male-sterile (CMS) 514A (2n=34) × (H.
angustifolius × P21, amphiploid) (2n=68)
3. F1 progeny of nuclear male-sterile (NMS) HA 89 (2n=34) × H. nuttallii (2n=34)
4. Oneamphiploid(2n=67)ofNMSHA89×H.maximiliani(2n=34)

44.  Procedure:
Interspecific hybrids and backcross progenies involving four wild perennial
species, H. californicus, H. angustifolius, H. nuttallii and H. maximiliani, were
examined.
 Genomic DNA of wild sunflower species was used as a probe.
 Genomic DNA of HA89 was used as blocking DNA.
 Result:
GISH procedure helps to identify the extra alien chromosome in the background
of the 34 cultivated line chromosomes.
Results suggested two genomes belong to H. annuus, and two from H.
maximiliani with one chromosome missing. This also indicated that tracking the H.
maximiliani chromosomes or chromosome segments while selecting for specific
traits.

45. GISH analysis of backcross progenies and interspecific hybrids. The genomic DNA of wild sunflower species was labeled with
digoxingenin-11-dUTP and detected with anti-dig-rhodamine (red). Chromosomes were counterstained with DAPI (blue). The alien
chromosome (A) or segment (B) identified in the backcross progenies of H. californicus × HA 410. The additional alien chromosome (C)
or translocations (D) detected in the progenies of CMS 514A × (H. angustifolius × P21, amphiploid). (E) The genome of H. nuttallii
detected in the F1 hybrid of nuclear male-sterile HA 89 × H. nuttallii. (F) The H. maximiliani chromosomes detected in the amphiploid of
nuclear male-sterile HA 89 × H. maximiliani. Bars=5 μm.

46.  Analyzing genome architecture
 Repetitive DNA sequences
 Characterization of genome
 Phylogenetic Analysis
 Analysis of somaclonal variations
 Detection of Alien Chromatin
 Detection of Chromosomal aberration
 Chromosome organization at interphase nuclei
 cenM-FISH
 Chromosome paintings & diagnostic cytogenetics

47.  Many genes are similar in most plants
and the ordering of these genes is
highly conserved across wide
taxonomic grouping. In wheat and
rice, linear order of markers in
linkage group is same.

48. Following are various classes of repetitive DNA sequence that have value in
chromosomes and Genome identification through FISH administration: -
 rRNA genes.
Tandem repeats.
Telomeric seq.
Centromeric seq.
Microsatellites.

49.  GISH permits characterization of the
genome and chromosomes of hybrid
plants, alloploid species and recombinant
breeding lines i.e., ancestry of species
can be elucidated.
 Multicolor FISH (using total genomic
DNA as probe) used for discriminating
each genome in natural or artificial
amphidiploids.  used to distinguish 3
genomes of hexaploid wheat.

50. 50
 GISH provides opportunities in phylogenetic and taxonomic studies for
determining and testing genomic relationship of wild and cultivated plants.
 Classified 11 diploid species of Allium into 5 types, A to E  based on
chromosome localization and distribution patterns of 5S rRNA genes by means
of FISH.

51. 51
 In tissue culture: As novel source of
genetic variation of crop
improvement. Tissue culture phases
may impose stress, and induce
chromosome breakage and DNA
transposition, leading to karyotyping
changes.
 Examination of chromosomes
distribution of 5S and 18S-26S rRNA
is useful in identifying the types of
genomic changes.

52. 52
 In interspecific & intergeneric crosses aim is to transfer desirable trait from
wild into cultivable species.
 In plant breeding program, alien chromosomes , chromosome segments, and
genes can be identified and characterized by GISH and FISH.

53. 53
 FISH can provide a rapid & accurate identification of
most common trisomics and sex chromosomes
abnormalities, structure abnormalities.
 In many polyploid species, there are intergenomic
translocations shown by GISH.
 Almost aberration observed during meiosis
 The advantage of Multicolour FISH demonstration
of structural aberration which can not be detected by
conventional staining technique

54. 54
 Simultaneous visualization of total
genomic and highly repeated DNA as
probe is also useful for investigation
chromosome organization in interphase
nuclei, orientation of telomeres and
centromere.
 Plant telomeric sequence have been
cloned from Arabidopsis thaliana.

55. 55
 Centromere specific multicolor FISH is
a new technique that allows the
simultaneous characterization of all
human centromeres by using labelled
centromeric satellite DNA probe.

56. 56
 By mixing combinations of five fluors and
using special imaging software, can
distinguish all 23 chromosomes by
chromosome specific colors
 Whole chromosome or region specific
paints can be produced by interspersed
repetitive elements.
 Chromosome specific probes are now
commercially available for all human
chromosome that can be used in prenatal
and diagnostic cytogenetics.

57. FISH
GISH 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
 Relativelly expensive
 Closely related genomes in certain allopolyploids cannot be discriminate using GISH
technique
 mFISH is less sensitive and shows a lower degree of detection