Various exogenous and endogenous factors, such as environmental pollutants, ionizing radiation, drug chemotherapy, and cell metabolism, can cause various forms of DNA damage. Among them, DNA double-strand breaks have the most serious damage to genome stability and can threaten the survival of cells. Establishing a simple, rapid and sensitive method for detecting DNA hybridization and genotoxicity effects is a very meaningful research topic. Here is a brief introduction to the method of DNA damage detection.
What are the types of DNA damage detection
DNA damage detection methods include gel electrophoresis, ultraviolet spectrophotometry, fluorescence and electrochemistry, and chemiluminescence analysis. Chemiluminescence analysis (CL) has been widely used in the fields of environment and life sciences due to its high sensitivity, wide linear range, convenient operation, fast analysis, and easy automation. Formaldehyde and acetaldehyde are both environmental pollutants. To a certain extent, it can cause DNA damage. Study the amount of acridinium esters embedded in DNA before and after the damage of formaldehyde and acetaldehyde, causing changes in the chemiluminescence intensity of acridinium esters, and study the damage behavior of formaldehyde and acetaldehyde on DNA. Establish a simple chemiluminescence analysis method to evaluate the degree of DNA damage caused by formaldehyde and acetaldehyde.
Acridine esters method for detecting environmental damage to DNA
1. First, soak the glass luminescence cell used in a certain amount of concentrated nitric acid for several hours, acidify, and then rinse with water. Add 20 μL of 1mg/mL calf thymus DNA to the acidified luminescence cell, and place it for 1 hour to make The DNA is adsorbed on the bottom of the light-emitting pool, and then the unadsorbed calf thymus DNA is washed with secondary water to obtain the light-emitting pool with DNA adsorbed.
2. Add 50μL of 9.6×10-7g/mL acridinium ester to the luminescent cell, and the chimerization reaction is for 1h to embed the AE in the DNA double-stranded structure, and wash away the unchimeric AE with secondary water. Finally, in the luminescent cell Add luminescence initiation reagents in sequence to detect the chemiluminescence generated by AE chimerized in DNA. When detecting the damage of calf thymus DNA by formaldehyde and acetaldehyde, add damage reagent to the DNA after AE chimerization, and use it after a certain period of time. The second water washes away the AE released after the DNA is damaged, and the luminescence initiation reagent is added to detect the chemiluminescence intensity of the AE chimeric in the DNA.
Studies have shown that acridinium ester can be used as a chemiluminescence indicator with a good structure of intercalating DNA double helix. This detection method is feasible for DNA break damage and chemiluminescence detection method. It has the advantages of simplicity and sensitivity. Damage studies provide a simple research method. Desheng acridine ester luminescent markers have the properties of good hydrolytic stability, thermal stability and high signal-to-noise ratio, and the labeling conditions are mild, the labeling rate is high, and the separation does not affect the separation after labeling. , So it is considered to be an ideal chemical marker.
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