Fluorescence in situ hybridization originated in the late 1980s, which is a non-radioactive in situ hybridization developed on the basis of the original radioactive in situ hybridization. At present, in situ hybridization has been widely used in the study of chromosome structure, gene localization, tumor disease detection, environmental microbial detection and other fields.
Basic Principles: After the nucleotide probe labeled with biotin, digoxin and other reporter molecules and the homologous complementary target DNA on the detected chromosome or DNA fiber slice, the hybridization of nucleic acid probe and target DNA is formed through denature-anneal-renaturation. After that, the immunochemical reaction between the reporter molecule and the luciferase labeled specific avidin is utilized. The DNA was analyzed qualitatively, quantitatively or relatively-under microscope by fluorescence detection system.
Compared with traditional radiogenic in situ hybridization, fluorescence in situ hybridization has the following advantages:
- High sensitivity, high specificity, accurate localization. FISH probes can locate DNA sequences up to 1KB in length, which is comparable to the sensitivity of radioactive probes.
- Fluorescent probes are cost-effective, safe, stable, fast and have a short detection cycle.
- Can display a variety of colors at the same time, for a variety of sequences at the same time detection.
- It can detect chromatin hybridization in metaphase and interphase of cell division.
Experimental steps:
Factors affecting fluorescence signal intensity:
- Cell ribosome content
- Permeability around the cell
- Target sequence accessibility
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