Optimization of mtDNA purification from potato tu-bers using various sorption carriers and different de-grees of mitochondria isolation

Abstract

Currently, there is interest in assessing mtDNA damage as a marker of oxidative stress, and this is-sue is not sufficiently studied for plant mtDNA. However, the mitochondrial genome of plants has a number of features that can significantly complicate PCR. With insufficient purification of mitochondria, samples can still be contaminated with nuclear and plastid DNA, which can lead to non-specific annealing of primers. The aim of this work is to optimize the process of mtDNA isolation from potatoes (Solanum tuberosum L.) using various sorption media. The use of sorption methods on solid-phase carriers is most effective for large-sized mtDNA molecules. We compared the effectiveness of three commercial DNA isolation kits: diaGene (Dia-M, Russia), Proba-GS (DNA Technology, Russia) and Plasmid miniPrep (Evrogen, Russia). These methods involve binding DNA to the surface of sorption columns (diaGene, Plasmid miniPrep), or on a silicate sorbent (Proba-GS). To assess the effectiveness of the considered methods, the level of mtDNA saturation was measured using quantitative PCR. A comparison of the efficiency of DNA extraction at different degrees of purification of the initial material is also given – when using potato homogenate, when cleaning by differ-ential and centrifugation in the Percoll gradient. A low level of mtDNA saturation was observed when isolat-ed from the homogenate, and a high level was observed when mitochondria were isolated by gradient cen-trifugation. When using gradient centrifugation, a high level of mtDNA saturation was observed only when DNA was isolated by a set of Proba-GS. We found that the greatest saturation of the mtDNA material will be when using the Plasmid miniPrep kit during pre-purification of mitochondria by gradient centrifugation. On-ly gradient centrifugation can maximally purify the mitochondria from impurities. The use of the Plasmid miniPrep kit, which is designed for the isolation of plasmid DNA, also shows its effectiveness for mtDNA, which is similar to plastid DNA and is predominantly circular. As a result, it was possible to develop an op-timized process for isolating potato mtDNA, which is suitable for further molecular genetic studies. This method can be used for any scientific research related to the study of the mitochondrial genome of plants.