Insitu Hybridization......in crop plants

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In Situ Hybridization A.K. Chhabra Jitender DISCLAIMER All copyrights of the figures used in the present presentation lie with the original developers, The information has been gathered here for educational purpose and not for sale.

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Uses

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Procedure

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Tissue In situ hybridization

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In situ hybridization by mRNA mRNA in situ hybridization detection using tyramide signal amplification (TSA). In the presence of horseradish peroxidase (HRP) and hydrogen peroxide, tyramide radicals are formed (red box) that can covalently react with nearby residues.

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In Situ Hybridization Conclusions: In early stages, transcript is distributed throughout the embryo. As the embryo matures, transcript levels remain high but begin to concentrate in the developing nervous system. In the mature embryo, gene expression is restricted to the specific region. Warning: In situ is a qualitative process to visualize gene expression. Do not use it quantitatively. What does it look like?

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Permeabilization problems or difficulty getting probes into tough cells like Gram+, Archaea, etc. Uneven cell penetration No signals due to metabolically inactive or quiescent cells High amount of background autofluorescence including cyanobacteria, plant tissues, etc Decreased amounts of rRNA target low or no ribosomes PROBLEMS WITH IN SITU HYBRIDIZATION

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Fluorescence in situ Hybridization Developed by Prof. Dr. R. Amann FISH involves the preparation of short sequences of single-stranded DNA, called probes, which are complementary to the DNA sequences the researchers wish to paint and examine. These probes hybridize, or bind, to the complementary DNA and, because they are labeled with fluorescent tags, allow researchers to see the location of those sequences of DNA. Unlike most other techniques used to study chromosomes, which require that the cells be actively dividing, FISH can also be performed on non-dividing cells, making it a highly versatile procedure.

Fluorescence in situ Hybridization (FISH) : 

Fluorescence in situ Hybridization (FISH) FISH - a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules Detects nucleic acid sequences by a fluorescent probe that hybridizes specifically to its complementary target sequence within the intact cell FISH involves the use of short sequences of single-stranded DNA (probes) which are labeled with fluorescent tags, to hybridize, or bind, to the complementary DNA to see the location of those sequences of DNA under the fluorescent microscope FISH involves the preparation of short sequences of single-stranded DNA, called probes, which are complementary to the DNA sequences the researchers wish to paint and examine. These probes hybridize, or bind, to the complementary DNA and, because they are labeled with fluorescent tags, allow researchers to see the location of those sequences of DNA. Unlike most other techniques used to study chromosomes, which require that the cells be actively dividing, FISH can also be performed on non-dividing cells, making it a highly versatile procedure.

Uses :Fluorescence in situ Hybridization (FISH) : 

Uses :Fluorescence in situ Hybridization (FISH) Identifies chromosomal abnormalities. Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination. Identification of cultured/uncultured micro-organisms.

Uses: Fluorescence in situ Hybridization (FISH) : 

Uses: Fluorescence in situ Hybridization (FISH) Used to identify the presence and location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections. Often used during Metaphase but is now used on Interphase chromosomes as well.

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(FISH) Steps:

Procedure : 

Procedure

Fluorescence in situ Hybridization (FISH) Advantages: : 

Fluorescence in situ Hybridization (FISH) Advantages: Less labor-intensive method for confirming the presence of a DNA segment within an entire genome than other conventional methods like Southern blotting High efficiency of hybridization and detection Result may be easier to interpret than interpretation of complex karyotype

Fluorescence in situ Hybridization (FISH) Advantages: : 

No cell culture needed Can combine FISH with immunostaining i.e. FICTION technique Rapid and sensitive Lots of cells can be analyzed Fluorescence in situ Hybridization (FISH) Advantages:

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Fluorescence in situ Hybridization (FISH) Disadvantages: Need specialized camera and image capture system Limited number of commercial probes available FISH will only provide information about the probe being tested, other aberrations will not be detected

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Eubacteria DAPI

FISH Procedure : 

FISH Procedure

FISH Procedure : 

FISH Procedure Denature the chromosomes Denature the probe Hybridization Fluorescence staining Examine slides or store in the dark

FISH Uses : 

FISH Uses Detection of high concentrations of base pairs Eg: Mouse metaphase preparation stained with DAPI (a non-specific DNA binding dye with high affinity for A-T bonds)

Applications FISH : 

Applications FISH 1 Identifying/quantifying uncultivated and/or cultivated organisms in natural environmental samples, within organisms like endosymbionts, or enrichments. 2 Monitoring population dynamics in environments and/or enrichments. 3 Visualizing spatial relationships among microbes in communities like biofilms, sludge, etc. 4 Identifying morphology or relationships among types of organisms.

Williams Syndrome : 

Williams Syndrome The chromosomal deletion that causes Williams Syndrome is so small that it cannot be seen in a karyotype. However, the deletion can be observed using a special technique called fluorescent in situ hybridization, or FISH. http://learn.genetics.utah.edu/units/disorders/karyotype/williams.cfm

Example of FISH : 

Example of FISH this method is used to identify the bacterial community in the insects gut. Fluorescence labeled oligo-nucleotide probe are synthesized (1), which will recognize a specific RNA sequence in the ribosome (2). These sites are highly diverse between species. The species are distinguished by the emitted  light (3) of the specific fluorescent probes The samples are observed under a fluorescence microscope (4). www.ice.mpg.de/bol/equipment/fish.htm insect gut Insect gut

Example of FISH : 

Example of FISH Bacteria from the insect gut: Blue is stained with DAPI. Red is by CY3 labeled FISH probe. With the FISH probe, the bacteria can be distinguished from another. Department of Bioorganic Chemistry Equipment and Method Development

ADVANTAGE OVER ISOTOPIC TECHNIQES : 

ADVANTAGE OVER ISOTOPIC TECHNIQES

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PROBE A small piece of nucleic acid that has been labeled with a radioactive isotope, dye, or enzyme and is used to locate a complementary nucleotide sequence or gene on a DNA molecule.

FISH PROBES : 

FISH PROBES Gene/locus specific probes Used to detect the presence absence or location of a particular gene, both in metaphase and interphase cells Centromere probes Designed to hybridize to alpha satellite repeat regions in centromeres, fluoresce brightly due to large number of repeats in centromeres Useful for determining the number of copies of a particular chromosome Whole chromosome paint probes Made from flow-sorted or micro-dissected chromosomes Used to determine composition of marker chromosomes, confirm the presence of chromosome rearrangements

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TYPES OF FISH PROBES

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SKY or M-FISH Spectral Karyotyping (SKY) and Multiplex Fluorescence In Situ Hybridization (M-FISH) SKY and M-FISH are molecular cytogenetic techniques that permit the simultaneous visualization of all human (or mouse) chromosomes in different colors, considerably facilitating karyotype analysis. Chromosome-specific probe pools (chromosome painting probes) are generated from flow-sorted chromosomes, and then amplified and fluorescently labeled by degenerate oligonucleotide-primed polymerase chain reaction.

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Spectral Karyotyping (SKY)

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Spectral karyotype (SKY) analysis of bone marrow cell with two translocations

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SKY and M-FISH (comparison): Both SKY and M-FISH use a combinatorial labeling scheme with spectrally distinguishable fluorochromes, but employ different methods for detecting and discriminating the different combinations of fluorescence after in situ hybridization. In SKY, image acquisition is based on a combination of epifluorescence microscopy, charge-coupled device (CCD) imaging, and Fourier spectroscopy. This makes possible the measurement of the entire emission spectrum with a single exposure at all image points.

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In M-FISH, separate images are captured for each of the five fluorochromes using narrow bandpass microscope filters; these images are then combined by dedicated software. In both techniques, unique pseudo-colors are assigned to the chromosomes based on their specific fluorochrome signatures SKY and M-FISH have applications in screening genomes for chromosomal aberrations in human disease and animal models of human cancer by making possible the unambiguous identification of even complex and hidden chromosomal abnormalities.

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SKY/M-FISH uses :

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Comparative Genomic Hybridization (CGH) Comparative genomic hybridization (CGH) is a fluorescent molecular cytogenetic technique that identifies DNA gains, losses, and amplifications, mapping these variations to normal metaphase chromosomes. It is a powerful tool for screening chromosomal copy number changes in tumor genomes and has the advantage of analyzing entire genomes within a single experiment.

Applications:- : 

Applications:- It is particularly applicable to the study of tumors which do not yield sufficient metaphases for cytogenetic analysis and can be applied to fresh or frozen tissues, cell lines, and archival formalin-fixed paraffin-embedded samples. CGH is based on quantitative two-color fluorescence in situ hybridization. Equal amounts of differentially labeled tumor genomic DNA and normal reference DNA are mixed together and hybridized under conditions of Cot-1 DNA suppression to normal metaphase spreads.

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The labeled probes are detected with two different fluorochromes, e.g., FITC for tumor DNA and TRITC for the normal DNA. The difference in fluorescence intensities along the chromosomes in the reference metaphase spread are a reflection of the copy number changes of corresponding sequences in the tumor DNA. Applications:-

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CGH has the advantage of requiring only genomic tumor DNA, making it highly useful for cancer cytogenetics, circumventing the need for high quality tumor metaphase spreads. The ability to study archival material allows retrospective analysis which can correlate chromosomal aberrations with the clinical course. Since its introduction in1992, CGH has been applied to a broad variety of tumor types which have previously defied comprehensive cytogenetic analysis by traditional methods.

CGH success story: : 

CGH success story: Revealed consistent genetic imbalances and multiple amplification sites in carcinomas of the brain, colon, prostate, cervix, and breast. For instance, it identified chromosome 7 gain and chromosome 10 loss as landmark aberrations in glioblastomas, and specific gains of chromosomes 1, 8, 17, and 20 and loss of 13q and 17p in breast cancer. Found chromosomal aberrations in human leukemia, lymphoma, and solid tumors has identified non-random tumor and tumor-stage specific genetic changes. This information can guide positional cloning efforts. Become an important initial screening test for chromosomal gains and losses in solid tumor progression, and the results derived from these experiments can be applied to the development of more specific diagnostics.

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phases of the comparative genomic hybridization (CGH) experimental lifecycle

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Fig: Detection of DNA loss or gain.

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METAPHASE FISH DISEASE INVOLVED DI-GEORGE SYNDROME WILLAMS SYNDROME STEROID SULFATASE DEFICIENCY KALLMAN SYNDROME

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INTERPHASE FISH ADVANTAGEOUS OVER METAPHASE ANEUPLOID SCREEN TEST

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Summary Hybridization is due to complementarity of DNA strands. DNA can be labeled various ways Isotopic and non isotopic Hybridization can detect identical or similar sequences.

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Conclusion FISH using r-RNA targeted probes is the method of choice for all studies in which exact cell numbers and cellular locations need to be determined. Development & design of more r-RNA targeted probes for novel microorganisms in environment The methodology is being continuously improved so far however, microscopic analysis by FISH has not been automated sufficiently which would be desirable in many investigations Accurate quantification still remains a challenging task and study needs careful controls.

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THANKS