A method for single-cell gel electrophoresis of algal cells

Abstract

The invention discloses a method for single cell gel electrophoresis of algal cells. The method comprises the steps of preparation of a single cell gel electrophoresis reagent, manufacture of a micro electrophoresis bath, preparation of a green algal cell sample and improvement and optimization of single cell gel electrophoresis cells, wherein the micro electrophoresis bath is manufactured by adopting a common smooth glass slide, conducting surface modification on the glass slide, and laying only one layer of gel, and compared with the traditional method that three gel layers need to be laid on the glass slide for sample preparation and the follow-up improved double-layer gel process, the advantages of simple sample preparation process, high success rate, easy operation, low background fluorescence value, definite sample images, low experiment cost and the like are achieved. Bases on the characteristic that the green algal cells have cell walls, the conditions for cell disruption are improved; according to different types of DNA damage actions, corresponding endonucleases are added for enzymolysis, so that damaged nuclear DNAs in the green algal cells can be more easily migrated under the action of an electric field, and the experiment success rate is remarkably improved.

Description

technical field [0001] The invention relates to single-cell detection technology, and particularly provides a method for single-cell gel electrophoresis of algae cells. Background technique [0002] Single-cell gel electrophoresis, also known as the comet assay, is a technique first proposed by Ostling et al. in 1984 to identify genotoxicity by detecting DNA strand damage. It can effectively detect and quantitatively analyze the degree of DNA single- and double-strand nick damage in cells. When various endogenous and exogenous DNA damage factors induce cell DNA strand breaks, its superhelical structure is destroyed. and other components diffuse into the cell lysate, while nuclear DNA can only stay in place due to its large molecular weight. Under neutral conditions, DNA fragments can enter the gel to migrate, and under the action of alkaline electrolyte, DNA unwinds, and the damaged DNA chains and fragments are released. Due to the small molecular weight of these DNAs and...

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Problems solved by technology

et al speculated on this phenomenon, and believed that there are some technical difficulties in detecting DNA damage of Chlamydomonas reinhardtii by this method: (1) the existence of cell wall leads to the problem of low cell lysis efficiency; (2) there are physical damages around the nucleus. or biological barriers, so that the incidence of "comet" tailing phenomenon is low; (3) there are difficulties in "comet" image observation and DNA damage quantification ( et al.,2004)

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