Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, 162 06, Prague 6, Czech Republic.
Mol Biol Rep. 2019 Jun;46(3):3063-3072. doi: 10.1007/s11033-019-04742-6. Epub 2019 Mar 11.
The efficiency of solid phase extraction (SPE) of DNA on polymer particles is limited by the features of the applied solid support, such as size, hydrophilicity, and functionality and their application in SPE also requires additional steps and compounds to finally obtain sufficient amount of high-quality DNA. The present study describes a preparation of sub-micrometer monodisperse poly(methacrylic acid-co-ethylene dimethacrylate) (PME) particles by precipitation polymerization. The effect of the ethylene dimethacrylate (EDMA) crosslinker concentration on morphology and particle size, which varied from 730 to 900 nm, was investigated. The particles with 5 and 15 wt% EDMA were selected for a study of SPE of plasmid DNA under various adsorption and elution conditions, followed by the enzymatic restriction of isolated DNA to verify a quality the nucleic acid. The particles with 15 wt% EDMA were suitable for the SPE because they retained better colloidal stability during the adsorption without additional induction of DNA conformational change. The quality of isolated DNA was finally verified by enzymatic restriction by restriction endonuclease EcoRI. Moreover, the developed method using PME particles was successfully utilized for DNA isolation from Escherichia coli lysate.
固相萃取(SPE)中聚合物颗粒对 DNA 的萃取效率受到应用的固体支撑物的特性限制,如大小、亲水性、功能等,其在 SPE 中的应用也需要额外的步骤和化合物,以最终获得足够数量的高质量 DNA。本研究描述了一种通过沉淀聚合制备亚微米单分散聚(甲基丙烯酸-co- 乙二甲基丙烯酸酯)(PME)颗粒的方法。研究了乙二甲基丙烯酸酯(EDMA)交联剂浓度对形态和粒径的影响,粒径从 730 到 900nm 不等。选择具有 5wt%和 15wt%EDMA 的颗粒,在各种吸附和洗脱条件下用于质粒 DNA 的 SPE 研究,然后用酶对分离的 DNA 进行限制酶切,以验证核酸的质量。具有 15wt%EDMA 的颗粒适用于 SPE,因为它们在吸附过程中保持更好的胶体稳定性,而不会额外诱导 DNA 构象变化。最后通过限制性内切酶 EcoRI 的酶切来验证分离的 DNA 的质量。此外,还利用开发的 PME 颗粒方法成功地从大肠杆菌裂解物中分离 DNA。