The principle and application of fluorescence in situ hybridization technology

In situ hybridization (ISH) refers to the technique of hybridizing target DNA or RNA sequences labeled with radioactive or non radioactive substances with chromosomal DNA to detect the position and distribution characteristics of the target sequence on the chromosome. Fluorescence in situ hybridization (FISH) refers to in situ hybridization using fluorescent markers instead of radioactive isotope labeled probes. This article provides an overview of the principle of fluorescence in situ hybridization technology, selection of biomarkers, key technical points, development, and applications.

1、 Principles 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 form a hybrid of target DNA and nucleic acid probe through denaturation annealing renaturation. Labeling a certain nucleotide of a nucleic acid probe with a reporter molecule such as biotin or digoxin can utilize the immunochemical reaction between the reporter molecule and the specific avidin labeled with fluorescein, and perform qualitative, quantitative, or relative localization analysis of the tested DNA under the microscope using a fluorescence detection system. Compared with other hybridization techniques, fluorescence in situ hybridization (FISH) has some advantages: short cycle time, high stability, and very safe; High resolution, 3-20 Mb; The probe can be stored for a long time; Multi color markings, simple and intuitive; 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 and functional genomes.