Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic.
Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic.
Folia Biol (Praha). 2021;67(1):10-15. doi: 10.14712/fb2021067010010.
The crucial requirement of molecular genetic methods is high-quality input material. The key question is "how to preserve DNA during long-term storage." Biobanks are recommended to aliquot isolated DNA into provided volumes. The aim of this study was to analyse the effect of repeated freezing and thawing on the genomic DNA integrity, quality and concentration. The aliquoted DNA isolated from blood cells using the automatic MagNA system and manual salting out method underwent freeze/thaw cycles at different storage conditions (-20 °C, -80 °C and liquid nitrogen). The average initial concentrations were 270.6 ng/μl (salting out method) and 125.0 ng/μl (MagNA). All concentration deviations relative to the concentration after the first freeze/ thaw cycle were less than 5 % for -20 °C and -80 °C cycling with both isolation methods. The average percentage differences of liquid nitrogen samples were higher, and the MagNA isolation method showed significant differences. There were no significant changes in the DNA purity or quality. The repeating freeze/ thaw up to 100 cycles (through -20 °C and -80 °C, respectively) did not significantly influence the integrity, concentration, or purity of genomic DNA, suggesting that storage of samples in high-volume pools without multiple aliquoting is possible. Storage in a freezer seems to be the most suitable way of long-term DNA preservation, because liquid nitrogen storage leads to formation of DNA clumps.
分子遗传学方法的关键要求是高质量的输入材料。关键问题是“如何在长期储存过程中保存 DNA”。生物银行建议将分离的 DNA 按规定体积分装。本研究旨在分析反复冻融对基因组 DNA 完整性、质量和浓度的影响。使用自动 MagNA 系统和手动盐析法从血细胞中分离出的等分 DNA 在不同的储存条件（-20°C、-80°C 和液氮）下经历了冻融循环。初始平均浓度分别为 270.6ng/μl（盐析法）和 125.0ng/μl（MagNA）。对于两种分离方法，-20°C 和 -80°C 循环的所有浓度偏差相对于第一次冻融循环后的浓度均小于 5%。液氮样本的平均百分比差异更高，MagNA 分离方法显示出显著差异。DNA 纯度或质量没有明显变化。重复冻融至 100 次循环（分别通过-20°C 和-80°C）不会显著影响基因组 DNA 的完整性、浓度或纯度，这表明可以将样品储存在大容量池而无需多次等分。在冰箱中储存似乎是长期 DNA 保存的最佳方式，因为液氮储存会导致 DNA 聚集。
