PowerPoint Presentation: 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….. Probe...: 5 Probe... DNA or RNA seq. which is labeled and used to probe target nucleic acid. Classification Of Probe: 6 Classification Of Probe Nucleic acid type: Probe synthesis: Complexities of probe sequences : DNA RNA Cloned Synthetic PCR For repeated seq. For single copy For whole chr. For part of chr. For total genomic DNA Labelling Of Probes…: 7 Labelling Of Probes… Chemical labelling Enzymatic labelling Acetylaminofluorine Mercury Biotin Digoxigenin Labels…: 8 Labels… Radioactive labels Non-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 labelling procedures are of two types:- Direct ISH Indirect ISH Non-radioactive ISH procedures : 9 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 . Can be: - one step or two step (with 2 o reporter ) Non-radioactive Labels: 10 Non-radioactive Labels Biotin Digoxigenin Acetylaminofluorene Mercury Reporter Molecules in non-radioactive ISH: 11 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 ligands having an immunogenic group which can bind to same antibody. Signal Generating System: 12 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 Enzymes: 13 Enzymes Enzyme Substrate Color of ppt. Horseradish peroxidase Diamino benzidine(DAB) Red Alkaline phosphatase 5-Bromo 4-chloro 3-indolyl phosphate (BCIP) Blue Fluorochromes : 14 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 Multiple labeling : 15 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: 16 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 PowerPoint Presentation: 17 FISH & GISH FISH and GISH Techniques: 18 FISH and GISH Techniques In modification of in situ hyb. tech, A fluorescent molecule is deposited at the site of in situ hyb. , location of genes or DNA can be visualized on chromosomes. Fluorescence in situ hybridization (FISH) Total genomic DNA is used as probe in hyb. experiments Genomic in situ hybridization (GISH) Principles underlying in situ reactions: 19 Principles underlying in situ reactions Most of in situ reactions have a 1 o and 2 o step. Rex n s carried out on chromosomes in situ involve a 1 o rex n that defines the specificity of the rex n , and a 2 o rex n that provides the means for detecting the product of rex n . Chemicals used in preparation of chromosomes can determine the specificity of a primary reaction. Fluorescence in situ hybridization (FISH): 20 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 . FISH essentially involves seven steps:- : 21 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. Labeling of probe DNA: 22 Labeling of probe DNA The DNA to be used as probe has been inserted into MCS of plasmid. The whole recombined plasmid is labeled by nick translation method in presence of F-x-dUTP carrying fluorophore Fluorescein or Rhodamine. Nick translation produce random single strand breaks in double stranded DNA by pancreatic DNase-I . E.coli DNA polymerase I then catalyze the addition of nucleotide residue to the 3’ terminus of nick, with simultaneous removal of nucleotides from 5’ terminus. The nick moves linearly along DNA strand. PowerPoint Presentation: 23 Genomic in situ hybridization (GISH): 24 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…..: 25 GISH for plants….. was developed in 1987 by M.D. Bennett and J.S. Heslop-harrison GISH essentially involve eight steps: -: 26 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. PowerPoint Presentation: 27 Importance 1.Analyzing genome architecture through FISH: 28 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. Indicate that there are gross similarities in genome of two specie. 2. Repetitive DNA sequences: 29 2. Repetitive DNA sequences Following are various classes of repetitive DNA seq that have value in chromosomes and Genome identification through FISH administration: - rRNA genes. Tandem repeats. Telomeric seq. Centromeric seq. Microsatellites. 3. Characterization of genome: 30 3. Characterization of genome GISH permits characterization of the genome and chr. 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: 31 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 specie of Allium into 5 types, A to E based on chr. localization and distribution patterns of 5S rRNA genes by means of FISH. 5. Analysis of somaclonal variations: 32 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 chr. breakage and DNA transposition, leading to karyotyping changes. Examination of chr. distribution of 5S and 18S-26S rRNA is useful in identifying the types of genomic changes. 6. Detection of Alien Chromatin : 33 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 chr. , chr. Segments, and genes can be identified and characterized by GISH and FISH. 7.Detection of Chromosomal aberration: 34 7.Detection of Chromosomal aberration FISH can provide a rapid & accurate identification of most common trisomics and sex chr. abnormalities, structure abnormalities. In many polyploid species, there are intergenomic translocations shown by GISH. 8. Chromosome organization at interphase nuclei: 35 8. Chromosome organization at interphase nuclei Simultaneous visualization of total genomic and highly repeated DNA as probe is also useful for investigation chr. organization in interphase nuclei, orientation of telomeres and centromere & relationship b/w chromatin condensation and gene exp. Plant telomeric seq. have been cloned from Arabidopsis thaliana. 9. cenM-FISH: 36 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: 37 10. Chromosome paintings & diagnostic cytogenetics Whole chr. or region specific paints can be produced by interspersed repetitive elements. Chr. specific probes are now commercially available for all human chr. that can be used in prenatal and diagnostic cytogenetics References: -: 38 References: - Indian Journal of Biotechnology, Vol. 4, July 2005, pp. 305-315. Review Article- Fluorescence in situ hyb. in genome, chromosome and gene identification in plants. Chromosome biology- Rudi apples, Rosalind Morris, Bikram S. Gill, Cedric E. May In situ hyb. Notes by: - Dr. Satinder Kaur Molecular biology practical manual:- Ref by Dr. Parveen PowerPoint Presentation: 39 Queries ?