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1. FISH Technique (Fluorescent In Situ Hybridization)

2. Introduction Cytogenetics entered the molecular era with the introduction of in situ hybridization, a procedure that allows researchers to locate the positions of specific DNA sequences on chromosomes. Since the first in situ hybridization experiments in 1969 (Gall & Pardue, 1969), many variations of the procedure have been developed, and its sensitivity has increased enormously. Today, most in situ hybridization procedures use fluorescent probes to detect DNA sequences, and the process is commonly referred to as FISH (fluorescence in situ hybridization). A variety of FISH procedures are available to cytogeneticists, who use them to diagnose many types of chromosomal abnormalities in patients. The success of FISH, and all other methods of in situ hybridization, depends on the remarkable stability of the DNA double helix.

3. FISH FISH stands for Fluorescence In Situ Hybridization. Fluorescence in situ hybridization (FISH) is a laboratory technique for detecting and locating a specific DNA sequence on a chromosome. The technique relies on exposing chromosomes to a small DNA sequence called a probe that has a fluorescent molecule attached to it. The probe sequence binds to its corresponding sequence on the chromosome.

4. Principle of FISH The principle of FISH technique is HYBRIZATION . In molecular hybridization, a labeled DNA or RNA sequence is used as a probe to identify or quantify the naturally occurring counterpart of the sequence in a biological sample.

5. Steps of FISH The first step in the process is to make either a fluorescent copy of the probe sequence (Figure 1b, middle column) or a modified copy of the probe sequence that can be rendered fluorescent later in the procedure. Step 2. N ext, before any hybridization can occur, both the target and the probe sequences must be denatured with heat or chemicals. This denaturation step is necessary in order for new hydrogen bonds to form between the target and the probe during the subsequent hybridization step.

6. Step 3. Th e probe and target sequences are then mixed together , and the probe specifically hybridizes to its complementary sequence on the chromosome. Step 4. The next step is washing off extra probe using suaitable solvent. Step 5. H ybrids formed between the probes and their chromosomal targets can be detected using a fluorescent microscope.

7. (a) The basic elements of FISH are a DNA probe and a target sequence. (b) Before hybridization, the DNA probe is labeled by various means, such as nick translation, random primed labeling, and PCR. Two labeling strategies are commonly used: indirect labeling (left panel) and direct labeling (right panel). For indirect labeling, probes are labeled with modified nucleotides that contain a hapten, whereas direct labeling uses nucleotides that have been directly modified to contain a fluorophore. (c) The labeled probe and the target DNA are denatured. (d) Combining the denatured probe and target allows the annealing of complementary DNA sequences. (e) If the probe has been labeled indirectly, an extra step is required for visualization of the nonfluorescent hapten that uses an enzymatic or immunological detection system. Whereas FISH is faster with directly labeled probes, indirect labeling offers the advantage of signal amplification by using several layers of antibodies, and it might therefore produce a signal that is brighter compared with background levels.

8. Flourochromes used in FISH The DNA-specific fluorochromes such as 4′,6-diamidino-2-phenylindole (DAPI) One of the most common DNA stains is DAPI (4',6-diamidino-2-phenylindole) which binds to A-T rich regions of the DNA double helix. DAPI fluorescence intensity increases if attached to DNA compared to its unbound state. It is excited by UV-light with a maximum at 358 nm. Emission spectrum is broad and peaks at 461 nm Hoechst 33258 (H33258) -blue colour Mithramycin Fluorescein isothiocyanate

9. Types of probes One of the most important steps in FISH analysis is the choice of probe. A wide range of probes, extending from whole genomes to small cloned probes (1–10 kb), can be used. There are basically three types of probes, each with a different range of applications: 1.whole chromosome painting probes, 2.repetitive sequence probes, and 3. locus specific probes,

10. Uses of fish Karyotyping Detection of chromosomal abnormalities, Gene Mapping

11. Using FISH to Identify the Positions of Genes FISH provides a powerful tool for identifying the location of a cloned DNA sequence on metaphase chromosomes. Figure shows the results of a typical FISH experiment, in which a cloned DNA sequence was hybridized to normal metaphase chromosomes. Red bands are detected at hybridization sites on two homologous chromosomes, which can be identified by their characteristic banding patterns. Closer examination shows that each red band actually consists of two spots, corresponding to the two sister chromatids in a mitotic chromosome.

12. Diagnosing Chromosomal Abnormalities Using Karyotypes and FISH FISH and other in situ hybridization procedures are important in the clinical diagnosis of various chromosomal abnormalities, including deletions, duplications, and translocations. Investigators used FISH together with standard karyotyping to analyze a patient translocation. The hybridization probe corresponded to a segment of chromosome that was suspected to include the translocation breakpoint.

13. Using Collections of FISH Probes to “Paint” Entire Chromosomes cytogeneticists now have the option of using multifluor FISH, or spectral karyotyping, to quickly scan a set of metaphase chromosomes for potential rearrangements. Multifluor FISH generates a karyotype in which each chromosome appears to be painted with a different color. Each "paint" is actually a collection of hybridization probes for sequences that span the length of a particular chromosome.

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