Journal of Biochemistry Research

Fluorescence Insitu Hybridization

A variety of Fluorescence Insitu Hybridization 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. Shortly after Gall and Pardue's work, fluorescent labels quickly replaces radioactive labels in hybridization probes because of their greater stability, ease of detection and safety. In fact, most current in situ hybridization is done using Fluorescence Insitu Hybridization procedures (Trask, 2002; Speicher & Carter, 2005). Detecting a DNA sequence can be compared to looking for a needle in a haystack, with the needle being the DNA sequence of interest and the haystack being a set of chromosomes. This search is made much easier if the investigator has a powerful "magnet"—in this case, a fluorescent copy of the DNA sequence of interest. Hybridization takes place when the "magnet" unite the "needle"; this requires both a probe and a target. The probe sequence is often a piece of cloned DNA, is shown in red. The target DNA—chromosomes on a glass slide—is shown in blue. Hydrogen bonds that join the two strands of the DNA helix are represented by black lines. The Initial step in the process is to make either a fluorescent copy of the probe sequence  or a modified copy of the probe sequence that can be rendered fluorescent later in the procedure. Next, 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. The probe and target sequences are then mixed together, and the probe specifically hybridizes to its complementary sequence on the chromosome. If the probe is already fluorescent (middle column), it will be possible to detect the site of hybridization directly. In other cases (left column), an additional step may be needed to visualize the hybridized probe. Hybrids formed between the probes and their chromosomal targets can be detected using a fluorescent microscope.

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