Method for DNA conversion using sodium bisulfite with adsorption onto silica matrix
Abstract
Currently, studies related to DNA methylation are in focus of fundamental and applied research. They allow revealing unknown aspects of controlling cell proliferation and differentiation (including abnormal), understanding adaptation mechanisms, and developing methods for regulating the genome functioning without changing its nucleotide sequence. They also contribute to the development of methods for treating various tumours and other abnormalities associated with the disrupted expression of certain genes. The purpose of the work is to develop and optimise the method of DNA conversion with sodium bisulphite for further study of DNA methylation in eukaryotic cells. The method presented in this paper is based on DNA modification by deamination of unmethylated cytosine with sodium bisulphite. The procedure consists of four key stages: DNA denaturation with alkali, DNA modification by incubation with sodium bisulphite solution at high temperatures, DNA purification, and desulfonation. DNA purification is a critical stage, which was carried out by sorption of modified DNA on silicon dioxide. The solid-phase extraction made it possible to obtain modified DNA without bisulphite salt impurities, which was confirmed by the results of electrophoresis in 1% agarose gel in the presence of ethidium bromide. The described protocol allows for effective modification of DNA obtained from various tissues with a degree of DNA conversion of 96-98% with insignificant losses of the target product (33-41%) during the modification. This method optimises the process of bisulphite conversion by providing a reliable and cost-effective tool for analysing methylation patterns. It also contributes to a better understanding of the regulation of gene expression and the biology of carcinogenesis. It can also facilitate the production of organisms with desired properties.
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