Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX, 78666, USA.
Materials Science, Engineering and Commercialization Program, Texas State University, San Marcos, TX, 78666, USA.
Anal Biochem. 2021 Mar 1;616:114099. doi: 10.1016/j.ab.2020.114099. Epub 2020 Dec 31.
Precipitation of DNA is performed frequently in molecular biology laboratories for the purpose of purification and concentration of samples and also for transfer of DNA into cells. Metal ions are used to facilitate these processes, though their precise functions are not well characterized. In the current study we have investigated the precipitation of double-stranded DNA by group 1 and group 2 metal ions. Double-stranded DNAs were not sedimented efficiently by metals alone, even at high concentrations. Increasing the pH to 11 or higher caused strong DNA precipitation in the presence of the divalent group 2 metals magnesium, calcium, strontium and barium, but not group 1 metals. Group 2 sedimentation profiles were distinctly different from that of the transition metal zinc, which caused precipitation at pH 8. Analysis of DNAs recovered from precipitates formed with calcium revealed that structural integrity was retained and that sedimentation efficiency was largely size-independent above 400 bp. Several tests supported a model whereby single-stranded DNA regions formed by denaturation at high pH became bound by the divalent metal cations. Neutralization of negative surface charges reduced the repulsive forces between molecules, leading to formation of insoluble aggregates that could be further stabilized by cation bridging (ionic crosslinking).
DNA 的沉淀在分子生物学实验室中经常进行，目的是纯化和浓缩样品，以及将 DNA 转入细胞。金属离子被用于促进这些过程，尽管它们的确切功能尚未得到很好的描述。在本研究中，我们研究了一组 1 和一组 2 金属离子对双链 DNA 的沉淀作用。单独使用金属离子不能有效地沉淀双链 DNA，即使在高浓度下也是如此。将 pH 值提高到 11 或更高会导致在二价组 2 金属镁、钙、锶和钡存在下强烈沉淀 DNA，但组 1 金属则不会。组 2 的沉淀曲线与过渡金属锌明显不同，锌在 pH 值为 8 时就会引起沉淀。对用钙形成的沉淀物中回收的 DNA 的分析表明，结构完整性得以保留，并且在 400 bp 以上，沉淀效率基本上与大小无关。有几个测试支持了一个模型，即在高 pH 值下变性形成的单链 DNA 区域被二价金属阳离子结合。中和负表面电荷减少了分子之间的排斥力，导致形成不溶性聚集体，这些聚集体可以通过阳离子桥接（离子交联）进一步稳定。
