Preparation method of sample for conveniently and rapidly detecting deoxyribonucleic acid (DNA) cell damage and kit using same

Abstract

The invention discloses a preparation method of a sample for conveniently and rapidly detecting deoxyribonucleic acid (DNA) cell damage. The method mainly comprises a gel sheet-making step, a cell lysis step, a DNA melting step, an electrophoresis step, a neutralizing step and a dyeing step, wherein in the gel sheet-making step, two layers of gels need to be spread; and in the dyeing step, a GelRed nucleic acid gel dye with high sensitivity and low toxicity is used. The invention also provides a kit using the method. The kit comprises normal melting-point agarose, low melting-point agarose, a cell lysis solution, an electrophoretic buffer solution, the DNA gel dye and a frosted edgeglass slide. According to the method, fluorescence dyeing is carried out by using the GelRed nucleic acid gel dye, and the GelRed nucleic acid gel dye is high in sensitivity, low in toxicity and stable, and environment pollution cannot be caused by wastes, so that the method is safe and environment-friendly; and because of only two layers of the spread gels, compared with a sandwich gel-spreading method used in the traditional comet assay, the preparation method is easy to operate and difficult to degum, and is uniform in dyeing; and moreover, the obtained electrophoresis image is relatively clear and objective.

Description

technical field [0001] The invention relates to mammalian cell DNA damage detection technology, in particular to a method for preparing samples for comet experiment detection and a kit used in the method. Background technique [0002] DNA stores the genetic information that organisms rely on to survive and reproduce. Cellular DNA damage is one of the hotspots in the field of cell biology. When a cell is damaged, its DNA double strand will break, and a specific cascade of biochemical reactions can occur during the process, which depends on Ca 2+ , Mg 2+ The endonuclease is activated, preferentially acts on the internucleosome region connecting DNA, cuts the DNA chain into fragments of 180-200 bp or multiples thereof, and the migration speed and migration distance of DNA fragments in comet electrophoresis are several times The nuclear DNA of normal cells presents a "comet" shape detached from the nucleus, which can distinguish DNA damaged cells from normal cells. [0003] ...

AI Technical Summary

Problems solved by technology

Usually, the fluorescent dye used in the fluorescent staining step is Ethidium Bromide (EB), and EB is a strong mutagen, which is easy to volatilize into the air and pose a threat to the health of test operators; use Throwing the final stained slides directly into the trash can pose a potential hazard to the surrounding environment

In addition, the existing gel slides used for comet electrophoresis analysis require three layers of gel, and in the subsequent lysing, electrophoresis and rinsing processes, the gel is easily detached from the slide, resulting in gel failure and affecting comet electrophoresis. Promotion and Application of Technology

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