Keck Graduate Institute of Applied Life Sciences , 535 Watson Drive, Claremont, California 91711, United States.
J Phys Chem B. 2013 Sep 19;117(37):10742-9. doi: 10.1021/jp405753m. Epub 2013 Sep 4.
Solid phase extraction and purification of DNA from complex samples typically requires chaotropic salts that can inhibit downstream polymerase amplification if carried into the elution buffer. Amino acid buffers may serve as a more compatible alternative for modulating the interaction between DNA and silica surfaces. We characterized DNA binding to silica surfaces, facilitated by representative amino acid buffers, and the subsequent elution of DNA from the silica surfaces. Through bulk depletion experiments, we found that more DNA adsorbs to silica particles out of positively compared to negatively charged amino acid buffers. Additionally, the type of the silica surface greatly influences the amount of DNA adsorbed and the final elution yield. Quartz crystal microbalance experiments with dissipation monitoring (QCM-D) revealed multiphasic DNA adsorption out of stronger adsorbing conditions such as arginine, glycine, and glutamine, with DNA more rigidly bound during the early stages of the adsorption process. The DNA film adsorbed out of glutamate was more flexible and uniform throughout the adsorption process. QCM-D characterization of DNA elution from the silica surface indicates an uptake in water mass during the initial stage of DNA elution for the stronger adsorbing conditions, which suggests that for these conditions the DNA film is partly dehydrated during the prior adsorption process. Overall, several positively charged and polar neutral amino acid buffers show promise as an alternative to methods based on chaotropic salts for solid phase DNA extraction.
从复杂样品中固相提取和纯化 DNA 通常需要使用离液盐,如果将其带入洗脱缓冲液中,可能会抑制下游聚合酶扩增。氨基酸缓冲液可能是一种更兼容的替代方法,可以调节 DNA 与硅胶表面之间的相互作用。我们对氨基酸缓冲液促进的 DNA 与硅胶表面的结合以及随后 DNA 从硅胶表面的洗脱进行了表征。通过批量耗尽实验,我们发现与带负电荷的氨基酸缓冲液相比,更多的 DNA 从带正电荷的氨基酸缓冲液中吸附到硅胶颗粒上。此外,硅胶表面的类型极大地影响吸附的 DNA 量和最终的洗脱产率。利用耗散监测的石英晶体微天平实验(QCM-D)表明,在吸附条件较强的情况下,如精氨酸、甘氨酸和谷氨酰胺,会发生多相 DNA 吸附,在吸附过程的早期,DNA 会更牢固地结合。在谷氨酸中吸附的 DNA 膜在整个吸附过程中更加柔韧和均匀。从硅胶表面洗脱 DNA 的 QCM-D 表征表明,在较强吸附条件下,DNA 洗脱的初始阶段会吸收更多的水质量,这表明对于这些条件,在先前的吸附过程中,DNA 膜部分脱水。总的来说,几种带正电荷和极性中性的氨基酸缓冲液有望替代基于离液盐的固相 DNA 提取方法。