View by fluorescence microscope
View by fluorescence microscope
FIGURE 21.34 Fluorescence in Situ Hybridization—Principle
A cell with intact DNA in its nucleus is treated to denature the DNA, forming single-stranded regions. The fluorescently labeled DNA probe is added, and the single-stranded probe can anneal with the corresponding sequence inside the nucleus. The hybrid molecule will fluoresce when the light from a fluorescence microscope excites the tag on the probe. This technique can localize the gene of interest to different areas of the nucleus or to individual chromosomes.
practice, it is rarely necessary to use the whole gene sequence as a probe, unless distinguishing between closely related genes is essential. As the name indicates, the DNA probe is labeled with a fluorescent dye whose localization will eventually be observed under a fluorescence microscope. The tissue or cell must also be treated to denature the chromosomal DNA, but this is done on the actual tissue section, leaving the DNA within the nuclei.
A thin section of tissue from a particular animal, a mouse for example, may be treated with a DNA probe for a known mouse gene. In this case, the mouse probe will hybridize to the mouse DNA in the nucleus of all the cells. This tells us that the genes are in the nucleus, which we knew anyway. Some more useful applications are as follows:
1. Using a virus gene as a probe reveals which cells contain virus genes, and whether the virus genes are in the cytoplasm or have penetrated the nucleus.
2. Besides DNA within the nucleus, FISH can be used to identify where a particular gene is in the metaphase chromosome. First, a chromosome smear can be made on a microscope slide, and then probed with a fluorescently labeled gene of interest. The place where the probe binds reveals which chromosome carries the gene corresponding to the probe. With sufficiently sophisticated equipment, the gene may be localized to a specific region on the chromosome (Fig. 21.35).
3. A DNA probe can be used to detect mRNA within the target tissue since one of the two strands of the denatured DNA will bind to the RNA. Since mRNA is already single-stranded, the cells do not have to be treated with high heat or chemical denaturants. Cells actively transcribing the gene of interest will have high levels of the corresponding mRNA, which will bind the probe and light up (Fig. 21.36). The greater the gene expression, the brighter the cell will fluoresce. Identifying the location of a particular mRNA can be real helpful when comparing tissues. For example, comparing the amount of mRNA for a particular gene in liver cells versus heart cells can help determine the function of the gene of interest. Levels of many mRNA molecules are low and hence would give only a weak signal by FISH. In practice, such mRNA is often amplified by RT-PCR before detection (see Ch. 23). Alternatively, more modern and more sensitive techniques, such as microarrays are used for mRNA detection (see Ch. 25).
FISH can localize a gene to a specific place on a chromosome. First, metaphase chromosomes are isolated and attached to a microscope slide. The chromosomal DNA is denatured into single-stranded pieces that remain attached to the slide. The fluorescent probe hybridizes to the corresponding gene. When the slide is illuminated, the hybrid molecules fluoresce and reveal the location of the gene of interest.
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