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Fluorescence in-situ hybridization (FISH) is a molecular diagnostic technique that allows visualization of definite chromosome nucleic acid sequences within a cellular preparation.it involves use of precise annealing of fluorescence labeled DNA probe to complementary targeted sequences. Thus, the genes/sequence of interest could be observed visually using a fluorescence microscope. The most basic components of FISH are targeted DNA sequence & DNA probe. Before the hybridization can happen, the DNA probe has to be labeled by using various means for example: random primed labeling, nick translation or polymerase chain reaction. Two different strategies can be used for labeling: direct method & indirect method.
In indirect labeling, DNA probe is labeled with altered nucleotide that contains a hapten, while indirect labeling nucleotides that have been directly altered to contain a fluorophore are used. These fluorescently labeled probes, i. e., FISH probes are denatured first and subsequently, Combination of the denatured probe & target sequence allows the complementary DNA sequences to anneal. For the probes that have been labeled indirectly, an extra step is required to visualize the non-fluorescent hapten which often uses an enzymatic /immunological detection system. Although FISH is faster with the use of directly labeled probes, indirect labeling has the benefit of signal intensification by using numerous strata of antibodies, and thus, brighter signal production may take place as compared with background levels.
Due to its ability to detect chromosomal aberrations such as gene rearrangement, gene deletion and gene amplification & its high specificity; FISH procedures are widely used in molecular diagnostics to detect and identify:
FISH procedures are generally carried out on FFPE (Formalin-fixed paraffin embedded) tissue preparations or cellular preparations. Advantages of FISH analysis in comparison with conventional chromosomal analysis consist of the following:
Most of the FISH procedures are carried out in dark to avoid photo-bleaching(probes are highly sensitive to light). The FISH slides are observed under florescence microscope fitted with Excitation and Dichroic filters specific to probe. The FISH Procedure, as performed during internship is as follows:
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