The Fish Technique

The FISH technique is based on the ability of single-stranded DNA to anneal to complementary DNA. In the case of FISH, the target DNA is the nuclear DNA of interphase cells or the DNA of metaphase chromosomes that are affixed to a glass microscope slide. FISH can be accomplished with bone marrow or peripheral blood smears, or other fixed tissue, and modifications of FISH methodology have also permitted the analysis of nuclei extracted from paraffin-embedded tissues or frozen sections from solid tumors. The test probe is labeled by enzymatic incorporation of biotin- or digoxigenin-labeled nucleotides, or directly with fluorochromes, to produce fluorescent signals at the place where the target is located within the nucleus. The use of directly labeled probes has simplified the technique eliminating the probe detection

Fluorescent labelled probe

Target chromosomes on slides

Fluorescent labelled probe

Fish Labelled Probes

Fig. 1 Schematic illustration of the principles of FISH, showing two-color FISH for detection of two single locus sequences on metaphase chromosomes. (View this art in color at www.dekker.com.)

View on Fluorescent Microscope

Fig. 1 Schematic illustration of the principles of FISH, showing two-color FISH for detection of two single locus sequences on metaphase chromosomes. (View this art in color at www.dekker.com.)

steps. These probes have proven to yield strong signals and are suitable for clinical applications. The cellular DNA and labeled probe DNA are denatured by heating in a formamide solution to form single-stranded DNA. A solution containing the probe DNA is applied to the microscope slide, the slide is covered with coverslips and sealed, and hybridization is allowed to occur by overnight incubation at 37-40°C. Thereafter, the unbound probe is removed by extensive washes, and the slides are processed for probe detection.[3] The slides are typically counter-stained with DNA-binding fluorochromes, such as propi-dium iodide or 4,6-diamidino-2-phenylindole (DAPI). DAPI staining induces a chromosomal banding pattern that is identical to G-banding; thus DAPI is preferred for the analysis of metaphase cells (Fig. 1).

Probes for FISH

Several types of probes can be used to detect chromosomal abnormalities by FISH. These probes are usually divided into three groups: probes that hybridize to unique sequences; probes that hybridize to satellite DNA, tandem DNA repeats that are present at the centromeres of human chromosomes; and probes that hybridize to the whole chromosome, called, for this reason, painting probes. An increasing number of locus-specific probes have been established for the diagnosis of leukemias and lymphomas because genetic aberrations in these neo-plasias have been shown to predict clinical behavior and outcome better than morphology alone.[5] FISH assays for the detection of diagnostically relevant chromosomal changes in both myeloid and lymphoid neoplasias are commercially available from several companies (http:// www.vysis.com/,http://www.cytocell.co.uk/, and http:// www.qbiogene. com).

Fluorescence in situ hybridization methods have evolved dramatically during the last years leading to the development of multiple variants. Among those, single-and double-color FISH using satellite and locus-specific probes have special diagnostic applications. These probes can be applied to both metaphase and interphase cells and can detect various types of numerical or structural chromosomal aberrations. For the detection of translocations by conventional interphase FISH (I-FISH), differentially labeled probes flanking or spanning the respective breakpoints can be applied.[5]

To detect chromosomal translocations by I-FISH, there are mainly two types of probes depending on the nature of the involved genes. If both genes involved in a translocation are known, as in the t(9;22)(q34;q11) in chronic myeloid leukemia, differentially labeled probes spanning the respective genes are the most appropriate. The presence of a translocation leads to two fusion signals, one on each of the derivative chromosomes, in der(9)

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BCR22q11 ABL9q34

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Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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