FISH technology

Issuing time:2018-10-23 00:00

FISH technology

FISH(fluorescencein situ hybridization)Technique is an important non-radioactive in situ hybridization technique.The basic principle is that if the target DNA on the chromosome or DNA fiber section being tested is homologous and complementary to the nucleic acid probe being used,after denaturation-annealing-renaturation, target DNA and nucleic acid probe hybrids can be formed.

Introduction

FISH: fluorescence-labeled in situ hybridization

In 1974, Evans first combined chromosome banding with chromosome in situ hybridization to improve the accuracy of localization. In the late 1970s, people began to explore fluorescence labeling in situ hybridization, FISH technology.In 1981, Harper successfully located single-copy DNA sequences on G-banding specimens, which marked the important progress of chromosome mapping technique.In the 1990s, with the progress of the human genome project, FISH technology was rapidly developed due to the need to map the human genome with high resolution.Widely used.

Principle

A report molecule, such as biotin and digoxin, is labeled with a nucleotide of a nucleic acid probe.The chemical reaction of Phytophthora infestans was detected by fluorescence detection system under microscope.

Experimental flow

Preparation of Probe for FISH Sample Preparation of Probe Labeled Hybridization Chromosome and Analysis of Results by Fluorescence Microscopy.

Characteristic

The probes of in situ hybridization were classified into radioactive and non-radioactive labeling according to the types of labeled molecules.The advantage of radioprobes labeled with isotopes is that the sample preparation requirements are not high, and the signal intensity can be enhanced by extending the exposure time.Therefore, it is more sensitive.The disadvantages are unstable probe, long time of autoradiography, low spatial resolution due to radiation scattering, and complicated isotope operation.Using fluorescence labeling system can overcome these shortcomings, which is FISH technology.FISH as a non-radioactive detection system, has the following advantages: 1, fluorescent reagents and probes economic, safe;the probe is stable and can be used within two years after a mark;3, the experiment cycle is short, can get the result quickly, the specificity is good, the localization is accurate;fISH can locate DNA sequences with length of 1 KB, and its sensitivity is similar to that of radioactive probe.5. Multicolor FISH can detect multiple sequences simultaneously by displaying different colors in the same nucleus; 6. Changes in the number or structure of metaphase chromosomes can be shown on slides, and the structure of interphase chromosome DNA can be shown in suspension.

Disadvantages: can not achieve 100% hybridization, especially in the application of shorter cDNA probe efficiency decreased significantly.

Application

The technique can be used not only for the chromosomal localization of known genes or sequences, but also for the study of uncloned genes or genetic markers and chromosome aberrations. It has advantages in the research of gene characterization, quantification, integration and expression.


Share to:
地址:绍兴袍江群贤路与中兴大道东南角三楼310室