Comparison of sorption methods for total DNA isolation from muscle tissue: influence on the yield and integrity of mitochondrial DNA
Abstract
Neuromuscular diseases associated with genetic and mitochondrial impairments require high-quality DNA extraction from muscle tissue for subsequent molecular genetic analysis. This study presents a comparative evaluation of total DNA extraction methods from mouse skeletal muscle, including precipitation, silica membrane adsorption, bulk sorbents, and magnetic particles. The results demonstrated that although precipitation yielded the highest total DNA amount, the proportion of mitochondrial DNA (mtDNA) in these samples was minimal due to non-selective co-precipitation of degraded nuclear DNA fragments. In contrast, column-based adsorption and magnetic particle methods provided significantly higher mtDNA content (4.5-fold increase) and better preservation of mitochondrial genome integrity. Bulk sorbents showed the highest level of mtDNA damage, likely due to mechanical fragmentation and suboptimal elution conditions. Quantitative real-time PCR with SYBR Green proved to be the most effective method for assessing mtDNA integrity, enabling precise detection of even minor lesions, unlike qualitative approaches. Notably, SYBR Green did not inhibit amplification of ~2 kbp fragments, confirming its suitability for mtDNA analysis. The highest vulnerability to damage was observed in the mtDNA D-loop region, a key regulatory site, which is particularly relevant for studying mitochondrial dysfunction in muscle tissue. Thus, column-based or magnetic particle sorption methods combined with quantitative PCR are optimal for mtDNA studies, ensuring high sensitivity and reliability of results.
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Gao F., Schon K.R., Vandrovcova J., Wilson L., Hanna M.G. Ann Clin Transl Neu-rol. 2024; 12: 1680-1688. https://doi.org/10.1002/acn3.52141
Dubinin M.V., Mikheeva I.B., Stepano-va A.E., Igoshkina A.D., Cherepanova A.A., Semenova A.A., Sharapov V.A., Kireev I.I., Belosludtsev K.N. Biomolecules. 2024; 14: 316. https://doi.org/10.3390/biom14030316
Gureev A.P., Nesterova V.V., Sadovni-kova I.S. DNA Repair (Amst). 2025; 146: 103812. https://doi.org/10.1016/j.dnarep.2025.103812
Helbock H.J., Beckman K.B., Shigenaga M.K., Walter P.B., Woodall A.A., Yeo H.C., Ames B.N. Proc Natl Acad Sci U S A. 1998; 95: 288-293. https://doi.org/10.1073/pnas.95.1.288
Preetha Sh. American Journal of Bio-medical Science & Research. 2020; 8: 39-45. https://doi.org/10.34297/AJBSR.2020.08.001234
Patent № 5705628. DNA purification and isolation using magnetic particles. Haw-kins T. 1998. Whitehead Institute for Biomedi-cal Research, Cambridge. Mass.
Gureev A.P., Shaforostova E.A., Starkov A.A., Popov V.N. Toxicology. 2017; 382: 67-74. https://doi.org/10.1016/j.tox.2017.03.010.
Gureev A.P., Nesterova V.V., Sadovni-kova I.S. DNA Repair (Amst). 2025; 146; 103812. https://doi.org/10.1016/j.dnarep.2025.103812
Gonzalez-Hunt C.P., Thacker E.A., Tos-te C.M., Boularand S., Deprets S., Dubois L., Sanders L.H. Sci Rep. 2020; 10: 17293. https://doi.org/10.1038/s41598-020-74195-6
Gureev A.P., Andrianova N.V., Pevzner I.B., Zorova L.D., Chernyshova E.V., Sa-dovnikova I.S., Chistyakov D.V., Popkov V.A., Semenovich D.S., Babenko V.A., Sila-chev D.N., Zorov D.B., Plotnikov E.Y., Popov V.N. FEBS J. 2022; 289: 5697-5713. https://doi.org/10.1111/febs.16451
Meyer J.N. Ecotoxicology. 2010; 19: 804-811. https://doi.org/10.1007/s10646-009-0457-4
Hettiarachchi E., Grassian V.H. Lang-muir. 2024; 40: 27194-27205. https://doi.org/10.1021/acs.langmuir.4c02501
Karpov S.I., Selemenev V.F. In-frakrasnaya spektroskopiya sorbenkov: uchebnoe posobie / Voronezh : Izdatel`ko-poligraficheskij centr «Nauchnaya kniga», 2024. 376 p. (In Russ.)
Malik A.N., Czajka A. Mitochondrion. 2013; 13(5): 481-492. https://doi.org/10.1016/j.mito.2012.10.011
Mazunin I.O., Volod`ko N.V., Starikovskaya E.B., Sukernik R.I. Mole-kulyarnaya biologiya. 2010; 44(5): 755-772. (In Russ.)
Supruniuk E., Górski J., Chabowski A. Antioxidants (Basel). 2023; 12(2): 501. https://doi.org/10.3390/antiox12020501
Williams G.S.B., Boyman L., Chikando A.C., Khairallah R.J., Lederer W.J. Proc Natl Acad Sci U S A. 2013; 110(26): 10479-10486. https://doi.org/10.1073/pnas.1300410110
Caliendo A.M., Schuurman R., Yen-Lieberman B., Spector S.A., Andersen J., Man-jiry R., Crumpacker C., Lurain N.S., Erice A. J Clin Microbiol. 2001; 39(4): 1334-1338. https://doi.org/10.1128/JCM.39.4.1334-1338.2001
Lehle S., Hildebrand D.G., Merz B., Malak P.N., Becker M.S., Schmezer P., Ess-mann F., Schulze-Osthoff K., Rothfuss O. Nu-cleic Acids Research. 2014; 42: e41. https://doi.org/10.1093/nar/gkt1349.
Edwards J.G. Mitochondrion. 2009; 9: 31-35. https://doi.org/10.1016/j.mito.2008.11.004
