insitu hybridization

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Presentation Transcript

In situ hybridization : 

In situ hybridization Schematic representation of 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.

mRNA In Situ Hybridization : 

mRNA In Situ Hybridization Schematic diagram of the method employed in our ELF 97 mRNA In Situ Hybridization (E6604, E6605), Cytological Labeling (E6603) and Immunohistochemistry (E6600) Kits. Samples are probed with haptenylated or biotinylated target-specific probes such as antibodies or hybridization probes. Next, alkaline phosphatase conjugates of streptavidin or the hapten-specific probe are applied. Alternatively, a biotinylated antibody and biotinylated alkaline phosphatase can be used with standard bridging methods to increase the penetration in tissue, a method that is employed in our ELF 97 Immunohistochemistry Kit. The sample is then incubated with the ELF 97 phosphatase substrate, which forms an intense yellow-green–fluorescent ELF 97 alcohol precipitate at the site of alkaline phosphatase activity.

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In Situ Hybridization

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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 CNS. Warning: In situ is a qualitative process to visualize gene expression. Do not use it quantitatively. What does it look like?

Fluorescence in situ Hybridization : 

Fluorescence in situ Hybridization

Fluorescence in situ Hybridization (FISH) : 

Fluorescence in situ Hybridization (FISH) FISH - a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules Opening picture - Human M-phase spread using DAPI stain

Fluorescence in situ Hybridization (FISH) : 

Fluorescence in situ Hybridization (FISH) Identifies chromosomal abnormalities Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination

Fluorescence in situ Hybridization (FISH) : 

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

Fluorescence in situ Hybridization (FISH) : 

Fluorescence in situ Hybridization (FISH) This means you can view a segment or entire chromosome with your own eyes Was often used during M phase but is now used on I phase chromosomes as well

Fluorescence in situ Hybridization (FISH) : 

Fluorescence in situ Hybridization (FISH) Advantage: less labor-intensive method for confirming the presence of a DNA segment within an entire genome than other conventional methods like Southern blotting

FISH Procedure : 

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

FISH Procedure : 

FISH Procedure

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)

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SKY or M-FISH and CGH Techniques 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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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 The applications of SKY and M-FISH for screening genomes for chromosomal aberrations in human disease and animal models of human cancer are manifold. By making possible the unambiguous identification of even complex and hidden chromosomal abnormalities.

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SKY/M-FISH is particularly useful in : Mapping of chromosomal breakpoints Detection of subtle translocations Identification of marker chromosomes, homogeneously staining regions, and double minute chromosomes, Characterization of complex rearrangements.

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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.

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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.

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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 in 1992, CGH has been applied to a broad variety of tumor types which have previously defied comprehensive cytogenetic analysis by traditional methods.

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CGH has: 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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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. What is FISH?


FISH Fluorescent In Situ Hybridization A.K. Chhabra


FISH Fluorescent In Situ Hybridization Technique developed by Prof. Dr. R. Amann Ing. Reinhard AmannSalomon-Sulzer StrasseTechniker6845


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

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When a deletion is too small to see it in a karyotype, what do you do? Scientists have developed a more precise method than karyotype analysis to see the presence or absence of DNA sequences when looking at whole chromosomes. In this technique, a chemical that fluoresces (shines brightly) is first attached to a molecule that can recognize specific DNA sequences. This labeled molecule is called a "probe." Chromosomes are prepared in a way that allows the probe to bind to matching DNA sequences on the chromosomes. This process is called hybridization. The labeled DNA probe that is bound to the chromosomes fluoresces when the chromosomes are exposed to UV light, showing the presence and location of the probe-bound DNA sequences. If you can see fluorescence from the probe, the gene is present. If not, the gene has been deleted. FISH (Fluorescent In Situ Hybridization)

Illustration of the FISH technique : 

Illustration of the FISH technique FISH is used in many biological and medical research fields and has enabled to acquire a lot of knowledge concerning health, evolution, development, etc.


FISH FISH, PCR based techniques and conventional cytogenetics need to be considered together as all these methodologies can be used to detect numerical and structural cytogenetic aberrations. However, each method has its advantages and disadvantages and one may be superior to another in some circumstances, for example, FISH is rarely suitable for the detection of a specific translocation in a minimal residual disease setting whereas PCR is sensitive for the detection of low level disease. FISH is a relatively new molecular cytogenetics technology utilising fluorescently labelled DNA probes to detect or confirm gene or chromosome abnormalities that are generally either beyond the resolution of routine cytogenetics or can be applied to samples that are unsuitable for conventional cytogenetics analysis.

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.

Advantages & disadvantages of FISH Technique : 

Advantages & disadvantages of FISH Technique Main Advantages of FISH Techniques Direct FISH technique is relatively rapid and sensitive No cell culture needed Result may be easier to interpret than interpretation of complex karyotype Can combine FISH with immunostaining ie FICTION technique Main Disadvantages of FISH Techniques Need specialised 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

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). 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

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