The use of silicon dioxide as a sorbent for purification of DNA from rat liver tissue during bisulphite conversion
Abstract
Epigenetics is one of the most progressive areas in modern molecular biology. One of the mechanisms for the epigenetic regulation of gene activity is the process of DNA methylation. To determine the methyl status of a gene, DNA methylation analysis is most often used, accompanied by chemical modification of cytosine bases, followed by detection using methyl-specific PCR. Due to the fact that sodium bisulphite is a strong inhibitor of many enzymes, including DNA polymerase, with insufficient purification of the DNA preparation further amplification and detection become impossible. A modified method of DNA purification from sodium bisulphite using spin columns with silicon dioxide (SiO2) as a sorbent was used in the study. The object of the study was male laboratory rats (Rattus norwegicus L. of the Wistar line). Electrophoresis in 1% agarose gel allowed establishing that DNA purification from bisulphite using spin columns with silica gel showed a more effective result as compared to DNA treatment with an alcohol-glycerin mixture.
As a result of the quantitative assessment of DNA before and after bisulphite treatment, it was shown that the yield of DNA after spin column purification was 0.95 µg/µl (DNA concentration before treatment was 1.19 µg/µl), and losses were about 20%. Meanwhile the amount of DNA after purification from bisulphite by incubation of DNA in an ethanol-glycogen mixture was 0.14 µg/µl (losses of about 88%). As a result of the study, using horizontal electrophoresis in 2% agarose gel and further visualisation, PCR products of the following sizes were obtained: 518, 180, and 140 bp, losses of the target product during DNA modification with sodium bisulphite and subsequent purification were about 20%. The products obtained during methyl-specific PCR corresponded to the theoretically calculated size. It was shown that this modified method of DNA purification from sodium bisulphite is suitable for analysing the methyl status of the promoter of the pdhb pyruvate dehydrogenase gene when working with animal tissues, in particular with liver tissues of laboratory rats.
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References
Patkin E.L., Kvinn Dzh. E`pigeneticheskie mexanizmy` pre-draspolozhennosti k kompleksny`m patologiyam cheloveka. E`kol. genetika, 2010; 8(4): 44-56. (In Russ.)
Mehler M.F. Epigenetic principles and mechanisms underlying nervous sys-tem functions in health and disease. Progr. Neurobiol, 2008; 86: 305-341.
Darst R.P., Pardo C.E., Ai L., Brown K.D., Kladde M.P. Bisulfite se-quencing of DNA, Curr Protoc Mol Biol., 2010; 91(1): 7.9.1.-7.9.17. https://doi.org/10.1002/0471142727.mb0709s91
Fernandes Sara B., Grova Nathalie, Roth Sarah, Duca Radu Corneliu, Godderis Lode, Guebels Pauline, Mériaux Sophie B., Lumley Andrew I., Bouillaud-Kremarik Pascaline, Ernens Isabelle, Devaux Yvan, Schroeder Henri, Turner Jonathan D. N6-Methyladenine in Eukaryotic DNA: Tissue Distribution, Early Embryo Development, and Neuronal Toxicity, Front. Genet., 2021; 12: 657171. https://doi.org/10.3389/fgene.2021.657171
Ramalho-Carvalho J., Henrique R., Jerónimo C. Methylation-Specific PCR, Methods Mol Biol., 2018; 1708: 447-472. https://doi.org/10.1007/978-1-4939-7481-8_23
Rimola A., Costa D., Sodupe M., Lambert J-F., Ugliengo P. Silica Surface Features and Their Role in the Adsorption of Biomolecules: Computational Modeling and Experiments, Chem. Revs., 2013; 113(6): 4216-4313. https://doi.org/10.1021/cr3003054
Anokhina G.B., Selivanov A.Yu., Gryazev A.S., Eprintsev A.T., Chukhlebova O.E. Razrabotka effektivnogo metoda ochistki DNK pri bisul'fitnoi konversii s ispol'zovaniem oksida kremniya v kachestve sorbenta, Sorbtsionnye i khroma-tograficheskie protsessy, 2023; 23(2): 290-298. https://doi.org/10.17308/sorpchrom.2023.23/11152
Koptyaeva K.E., Muzhikyan A.A., Gushchin Ya.A., Belyaeva E.V., Makarova M.N., Makarov V.G. Metodika vskrytiya i izvlecheniya organov laboratornykh zhivotnykh (krysy), Laboratornye zhivotnye dlya nauchnykh issledovanii, 2018; (2). https://doi.org/10.29296/2618723X-2018-02-08
Rae Peter M.M., Barnett T.R., Bab-bitt D.G. Factors influencing the yield of satellite DNA in extractions from Drosoph-ila virilis and Drosophila melanogaster adults and embryos, Biochimica et Bio-physica Acta, 1976; 432(2): 154-160. https://doi.org/10.1016/0005-2787(76)90157-X
Moss D., Harbison S.A., Saul D.J. An easily automated, closed-tube forensic DNA extraction procedure using a thermo-stable proteinase, Int. J. Legal Med., 2003; 117(6): 340-349. https://doi.org/10.1007/s00414-003-0400-9
Wang R.Y.H., Gehrke C.W., Ehr-lich M. Comparison of bisulfite modifica-tion of 5-methyldeoxycytidine and deox-ycytidine residues. Nucleic Acids Res, 1980; 8(20): 4777-4790. https://doi.org/10.1093/nar/8.20.4777
Pajares M.J., Palanca-Ballester C., Urtasun R., Alemany-Cosme E., Lahoz A., Sandoval J. Methods for analysis of specif-ic DNA methylation status. Methods. Else-vier Inc., 2020; 187:3-12. https://doi.org/10.1016/j.ymeth.2020.06.021
