Fluorescence in situ hybridization (FISH) is a molecular diagnostic technique that allows the visualization of defined sequences of chromosomal nucleic acids within a cellular preparation. It involves the use of precise annealing of the fluorescently labeled DNA probe to complementary targeted sequences. Therefore, genes/sequences of interest could be observed visually using a fluorescence microscope. The most basic components of FISH are the targeted DNA sequence and the DNA probe. Before hybridization can occur, the DNA probe must be labeled using various means, for example: random primer labeling, nick translation, or polymerase chain reaction. You can use two different strategies for labeling: direct method and indirect method. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original assay In indirect labeling, the DNA probe is labeled with an altered nucleotide that contains a hapten, while in indirect labeling nucleotides that have been directly altered to contain a fluorophore are used. These fluorescently labeled probes, i.e. FISH probes, are first denatured and then. The combination of the denatured probe and the target sequence allows for annealing of complementary DNA sequences. For indirectly labeled probes, an additional 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 advantage of intensifying the signal using numerous layers of antibodies and, therefore, brighter signal production compared to background levels can occur. Thanks to its ability to detect chromosomal aberrations such as gene rearrangement, gene deletion and gene amplification and its high specificity; FISH procedures are widely used in molecular diagnostics to detect and identify: Centromeres of a specific chromosome. Specific oncogenes (locus-specific probe, useful for (ROS1,ALK), amplifications (HER-2), deletions (CLL) etc.). Specific tumor suppressor genes (probe locus specific, loss is relevant for tumor progression) Whole chromosomes (useful for detection of complex chromosomal rearrangements). FISH procedures are generally performed on FFPE (formalin-fixed paraffin) tissue preparations or cell preparations. The advantages of FISH analysis over conventional chromosome analysis are as follows: Please note: This is only one sample. Get a custom paper from our expert writers now. Get a custom assay Large numbers of cells can be examined (useful for detecting residual disease) Metaphase cells are not essential, so aberrations can be detected even in non-dividing cells (useful in chronic lymphocytic leukemia) FISH can be performed in a rather short period of time. Aberrations that are too subtle to be detected by the use of conventional cytogenetic analysis can also be identified. Most FISH procedures are performed in the dark to avoid photobleaching (the probes are highly sensitive to light). FISH slides are viewed under a fluorescence microscope equipped with probe-specific excitation and dichroic filters. The FISH procedure, performed during the internship, is as follows: Incubate the FFPE (formalin-fixed paraffin-embedded tissue) at 700°C for 3 hours. followed by deparaffinization in xylene.