Development of an effective method for DNA purification during bisulphite conversion using silicon oxide as a sorbent
Abstract
DNA methylation is one of the most studied mechanisms of epigenetic regulation of the genome, which controls gene expression in plants and animals. The development of effective methods for analysing the methyl status of genes is of great importance, since DNA treatment with sodium bisulphite is a multi-stage and labour-intensive process in which significant losses of the target product are unacceptable. The purity of the modified DNA plays a decisive role in the subsequent amplification process. The presence of high concentrations of various salts and impurities in the studied samples can inhibit the polymerase activity of DNA polymerase. In typical protocols describing the method of DNA conversion with sodium bisulphite, commercial kits are used for the purification of modified DNA, but their use often leads to large losses of the target product, which significantly reduces the efficiency of the purification process. In addition, due to large volume of analysed samples, the use of commercial kits significantly increases the cost of work. As part of the study, an effective, simple and inexpensive method was developed for purification of modified DNA from salts by sorption on silicon dioxide (SiO2). The use of finely dispersed silicon dioxide as a sorbent is due to its ability to selectively bind negatively charged DNA molecules. Analysis of the converted and purified DNA by electrophoresis in 2% agarose gel allowed to establish that the loss of the target product during the multistage treatment with sodium bisulphite was about 33%. The methyl-specific PCR using primers to individual CpG dinucleotides in the promoter of the GDH2 gene of maize (Zea mays L.) glutamate dehydrogenase demonstrated that the obtained amplicons corresponded to the theoretical size – 175 bp, 203 bp and 275 bp to the first, second and third analysed CpG dinucleotide, respectively. Therefore, it was shown that the matrix converted and adsorbed on silicon dioxide is suitable for the analysis of the DNA methyl status.
Downloads
References
Ashapkin V.V., Kutueva L.I., Aleksandrushkina N.I., Vanyushin B.F. Epigenetic mechanisms of plant adaptation to biotic and abiotic stresses. International Journal of Molecular Sciences. 2020; 21(20): 7457.
Vanyushin B.F., Ashapkin V.V. DNA methylation in higher plants: past, present and future. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms. 2011; 1809(8): 360-368.
Kirnos M.D., Aleksandrushkina N.I., Vanyushin B.F. 5-metiltsitozin v pirimidinovykh posledovatel'nostyakh DNK rastenii i zhivotnykh: spetsifichnost' metilirovaniya. Biochemistry. 1981; 46(12): 1458-1474.
Smirnikhina S.A., Lavrov A.V. Metody otsenki metilirovaniya ostatkov tsitozina v DNK. Molekulyarnaya biologiya. 2009; 43(3): 387-391.
Ryabushkina N.A., Omasheva M.E., Galiakparov N.N. Spetsifika vydeleniya DNK iz rastitel'nykh ob"ektov. Biotekhnologiya. Teoriya i praktika. 2012; 2: 9-26. (In Russ.)
Eprintsev A.T., Popov V.N., Fedorin D.N. Identifikatsiya i issledovanie ekspressii genov. Voronezh: Izd-vo Voronezh. un-ta, 2008. 63 p. (In Russ.)
Selemenev V.F., Rudakova L.V., Rudakov O.B., Belanova N.A., Nazarova A.A. Fosfolipidy na fone prirodnyh matricz. Voronezh: Science Book. 2020. 318 p. (In Russ.)
Selemenev V.F., Rudakov O.B., Slavinskaya G.V., Drozdova N.V. Pigmenty pishhevykh proizvodstv (Melanoidiny). M: Delhi Print. 2008. 246 p. (In Russ.)
Hsieh C.L. Evidence that protein binding specifies sites of DNA demethylation. Molecular and cellular biology. 1999; 19(1): 46-56.
Katevatis C., Fan A., Klapperich C.M. Low concentration DNA extraction and recovery using a silica solid phase. PLoS One. 2017; 12(5): 1-14. https://doi.org/10.1371/journal.pone.0176848
Selemenev V.F., Oros G.Yu., Rudenko I.V., Stukalov O.I., Tsyurupa M.P., Davankov V.A. Sposob polucheniya nukleinovykh kislot i aminokislot iz avtolizatov pekarskikh drozhzhei. 1999.
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. Chemical Reviews. 2013; 113(6): 4216-4313. https://doi.org/10.1021/cr3003054
Poeckh T., Lopez S., Fuller A.O., Solomon M.J., Larson R.G. Adsorption and elution characteristics of nucleic acids on silica surfaces and their use in designing a miniaturized purification unit. Analytical Biochemistry. 2008; 373(2): 253-262. https://doi.org/10.1016/j.ab.2007.10.026
Kramer M.F., Coen D.M. Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Molecular Biology. 2011; CHAPTER: Unit15.10.
