In situ hybridization (ISH) refers to the technique of detecting the position and distribution characteristics of a target DNA or RNA sequence on a chromosome by hybridizing it with chromosomal DNA after being labeled with radioactive or non radioactive substances. Fluorescence in situ hybridization (FISH) refers to in situ hybridization using fluorescent markers instead of radiolabeled probes. This article provides an overview of the principle of fluorescence in situ hybridization technology, the selection of biomarkers, key technical points, development, and applications.
1、 Principle of fluorescence in situ hybridization technology
Fluorescence in situ hybridization (FISH) is an important non radioactive in situ hybridization technique. The basic principle is that if the target DNA on the detected chromosome or DNA fiber slice is homologous and complementary to the nucleic acid probe used, the two can be denatured annealed renaturated to form a hybrid of the target DNA and nucleic acid probe. Labeling a certain nucleotide of a nucleic acid probe with a reporter molecule such as biotin or digoxin can be used to perform qualitative, quantitative, or relative localization analysis of the DNA to be tested under a microscope using a fluorescence detection system based on the immune chemical reaction between the reporter molecule and a specific affinity element labeled with fluorescein. Compared with other hybridization techniques, fluorescence in situ hybridization (FISH) has some advantages: short cycle, high stability, and very safe; High resolution, 3-20 Mb; probes can be stored for a long time; Multi colored markings, simple and intuitive; Simultaneously observing the localization of several DNA probes on the same slide under a fluorescence microscope can directly obtain their relative sequences and positions, greatly accelerating the research on the localization of biological genomes and functional genomes.