Insitu hybridization

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Insitu hybridization


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Slide 1:

1 In situ hybridization- GISH, FISH its importance

In situ hybridization:

2 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.

Basic technique:

3 Basic technique 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.

Different types localized by ISH:

4 Different types localized by ISH DNA sequences :- RNA sequences : - Viral sequences : - Repetitive seq. Unique seq. Whole chr. or a part of chr. Whole genome Helps to study the spatial & temporal patters of gene expression… Forms the basis of diagnosis of several viral diseases…..

Labeling Of Probes…:

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

Non-radioactive ISH procedures :

7 Non-radioactive ISH procedures 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 .

Non-radioactive Labels:

8 Non-radioactive Labels Biotin Digoxigenin Acetylaminofluorene Mercury Reporter Molecules in non-radioactive ISH 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 ligand s having an immunogenic group which can bind to same antibody.

Signal Generating System:

9 Signal Generating System Fluorochromes Enzymes Metals They get excited by light of one wavelength and emit light of another wavelength which is observed as fluorescence of different colors. Enzymes work by catalyzing the precipitation of a visible product at hybridization site. Colloidal gold which is conjugated to antibodies. Can be visualized with both light and electron microscope


10 Enzymes Enzyme Substrate Color of ppt. Horseradish peroxidase Diamino benzidine (DAB) Red Alkaline phosphatase 5-Bromo 4-chloro 3-indolyl phosphate (BCIP) Blue

Fluorochromes :

11 Fluorochromes Fluorochrome Fluorescene Fluorescene isothiocyanate (FITC) Green Tetramethyl rhodamine isothiocyanate (TRITC) Red Texas red or sulphorhodamine Deep red Amino methyl coumarine acetic acid (AMCA) Blue

Multiple labeling :

12 Multiple labeling In multiple labeling more than one probe can be employed simultaneously on target nucleic acid. This is very important:- To determine the relationship of different sequences with respect to each other. To identify different chromosomes simultaneously. To identify different genomes simultaneously. Types: - Sequential multiple labeling Simultaneous multiple labeling- Indirect method Simultaneous multiple labeling- Direct method

Example- Detection by Biotin:

13 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. + + ssProbe with biotin Avidin with SGS as FITC ssDNA

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FISH and GISH Techniques:

15 FISH and GISH Techniques In 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)

Fluorescence in situ hybridization (FISH) :

Fluorescence 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. A technique used to detect the presence or absence and location of specific gene sequences 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

Principles of fluroscent in situ reactions:

17 Principles of fluroscent in situ reactions Schematic diagram for FISH technique

FISH essentially involves seven steps:- :

18 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.

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Phenotypic instability, Karyotypic analysis of meiosis in synthetic hybrid of Arabidopsis Tyagi et al ., 2000 19

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Objective: To identify and track the B chromosome-containing sperm cell within mature pollen and pollen tubes by using B chromosome-specific DNA sequence ( pZmBs ) Material used : B chromosome-specific probe ( Alfenito and Birchler , 1993) to determine which sperm of a pair contains the B chromosomes and thus is predisposed to fertilize the egg (Carlson, 1969). Result : FlSH of this sequence in mature pollen showed FlTC detection most often (78.8%) in only one sperm and the vegetative nucleus (Figure 3); two hybridization signals were typically found within each of the somatic root tip nuclei.

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In Situ Hybridizations of a B Chromosome-Specific Probe ( pZmBs ) in the Pollen, Pollen Tube, and Root Tip of Maize. (A) DAPI-stained pollen grain showing the two sperm nuclei (SN) and the vegetative nucleus (VN). Bar = 10 nm. (B) Same pollen grain as in (A), showing two FITC signals (unlabeled arrowheads), which represent the locations of the B chromosome probe. Bar = 10 urn. (C) Composite of the original images of (A) and (B), showing the localization of the B chromosome probe (yellow dots; arrowheads) in one sperm and the vegetative nucleus. Bar = 10 nm. (D) DAPI-stained pollen tube showing two sperm nuclei (SN). Bar = 10 urn. (E) Same pollen tube as in (D), showing an FITC signal (arrowhead). Bar = 10 urn. (F) Composite of the original images of (D) and (E), showing the B chromosome probe (yellow dot; arrowhead) in one of the two sperm nuclei. Bar = 10 urn. (G) DAPI-stained root tip nuclei (RN). Bar = 10 urn. (H) Same nuclei as in (G), showing the FITC signals (arrowheads) within four of the five nuclei. Bar = 10 nm. (I) Composite based on the original images of (G) and (H), showing the position of the B chromosome probes (yellow dots; arrowheads) within the somatic nuclei. Bar = 10 urn.

Genomic in situ hybridization (GISH):

23 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 essentially involve eight steps: -:

24 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. GISH for plants was developed in 1987 by M.D. Bennett and J.S. Heslop-harrison

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Objective : To combine the genomes of O. sativa , a cultivar and O. punctata , a wild species using asymmetrical protoplast fusion technique . Procedure : Somatic hybrid production Genome identification - Chromosome preparation Genomic in situ hybridization Result All hybrids obtained were sterile because of the different genomes and the fluctuation of chromosome numbers

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Genomic in situ hybridization results. a. Tricolor discrimination for 3 genomes (red - A genome, blue - B genome and green - C genome) b. DAPI counterstaining c. Fluorescent green color for C genome detection d. Fluorescent red color for A genome detection

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Painted chromosomes in various days of hybridization. a. One day b. Two days c . Three days d. Four days

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Objective : To develop a GISH procedure for identifying chromosomes or chromosome segments of wild species in the background of cultivated sunflower Procedure Interspecific hybrids and backcross progenies involving four wild perennial species, H. californicus , H. angustifolius , H. nuttallii and H. maximiliani , were examined. Result - GISH procedure helps to identify the extra alien chromosome in the background of the 34 cultivated line chromosomes (Fig.1C) - 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.

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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.

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Importance of In situ hybridization

1.Analyzing genome architecture through FISH:

32 1.Analyzing genome architecture through FISH 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.

2. Repetitive DNA sequences:

33 2. Repetitive DNA sequences 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.

3. Characterization of genome:

34 3. Characterization of genome GISH permits characterization of the genome and chromosomes of hybrid plants, alloploid species and recombinant breeding lines i.e., ancestry of specie 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.

4. Phylogenetic Analysis:

35 4. Phylogenetic Analysis 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.

5. Analysis of somaclonal variations:

36 5. Analysis of somaclonal variations 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.

6. Detection of Alien Chromatin :

37 6. Detection of Alien Chromatin 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.

7.Detection of Chromosomal aberration:

38 7.Detection of Chromosomal aberration 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.

8. Chromosome organization at interphase nuclei:

39 8. Chromosome organization at interphase nuclei 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.

9. cenM-FISH:

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

10. Chromosome paintings & diagnostic cytogenetics:

41 10. Chromosome paintings & diagnostic cytogenetics 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 .

Slide 42:

Thank you

Possible questions:

Possible questions 1. Briefly explain in situ hybridization. 2. What is FISH? Explain its principle. 3. Write the importance of in situ hybridization.

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