Department of Chemistry, Indian Institute of Technology Madras, Chennai, 600036, India.
Department of Chemistry, Indian Institute of Technology Madras, Chennai, 600036, India.
Anal Chim Acta. 2024 Sep 8;1321:343045. doi: 10.1016/j.aca.2024.343045. Epub 2024 Aug 2.
Developing an alternative and benign method for DNA extraction is imperative due to the high cost and potential harms associated with conventional techniques. Investigation of Ionic liquid (IL) as a solvent for DNA storage and stability revealed the ability of IL to assist DNA processes. IL-based aqueous biphasic system emerges as a comprehensive extraction platform capitalizing on the task-specificity of ILs and the wide applicability of ABS for biomolecule extractions. Therefore, it is beneficial to optimize an IL-based ABS specifically for DNA extraction, taking into account the fundamental interactions between the IL and DNA.
The primary objective was to design ABS consisting of Ammonium based ILs, and Potassium phosphate buffer as the salting-out agent for the partitioning of salmon sperm DNA. The analysis focused on optimizing biocompatible anions for the extraction. Moreover, the stability of the DNA in the IL rich phases was analysed to validate the method. The proposed process was then employed for extracting plasmid DNA from bacteria, demonstrating results comparable to those obtained with a commercially available kit. Further validation using agarose gel electrophoresis and transformation of the extracted DNA into E.coli were conducted, producing promising outcomes. Although there is room for improvement in terms of recovery of DNA and reusability of ABS, the described approach is comparable with the conventional one while being cost-effective, and showcases a noticeable and convincing link to eco-friendly processes.
There is limited literature on IL-based ABS for DNA extraction, and the existing studies predominantly concentrate on systems derived from Cholinium ILs. However, their high hydrophilicity limits the choice of the second-phase forming component to polymers for the formation of ABS. Ammonium ILs efficiently form biphasic systems with various available salting-out agents, and biocompatible anions are introduced to mitigate the toxicity of the ILs.
由于传统技术成本高且潜在危害大，因此开发替代且良性的 DNA 提取方法势在必行。研究离子液体（IL）作为 DNA 储存和稳定性的溶剂，揭示了 IL 辅助 DNA 过程的能力。基于 IL 的双水相体系作为一种综合提取平台，利用 IL 的任务特异性和 ABS 对生物分子提取的广泛适用性而出现。因此，考虑到 IL 与 DNA 之间的基本相互作用，专门针对 DNA 提取来优化基于 IL 的 ABS 是有益的。
主要目标是设计由铵基 IL 和磷酸钾缓冲液组成的 ABS，作为鲑鱼精子 DNA 分配的盐析剂。分析重点是优化用于提取的生物相容阴离子。此外，还分析了 DNA 在富含 IL 的相中稳定性，以验证该方法。然后将该方法用于从细菌中提取质粒 DNA，结果与市售试剂盒相当。进一步使用琼脂糖凝胶电泳进行验证，并将提取的 DNA 转化为大肠杆菌，结果令人满意。尽管在 DNA 回收率和 ABS 可重复使用性方面还有改进的空间，但所描述的方法与传统方法相当，同时具有成本效益，并展示了与环保工艺的明显且令人信服的联系。
关于基于 IL 的 ABS 用于 DNA 提取的文献有限，现有的研究主要集中在源自 Cholinium IL 的系统上。然而，它们的高亲水性限制了第二相形成成分的选择，只能选择聚合物来形成 ABS。铵基 IL 与各种可用的盐析剂高效形成双水相体系，并引入生物相容阴离子来减轻 IL 的毒性。
