Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts
Altered nucleic acid partitioning during phenol extraction or silica adsorption by guanidinium and potassium salts
复制标题
胍盐和钾盐在苯酚提取或二氧化硅吸附过程中改变核酸分配。
DOI：
10.1016/j.ab.2011.08.047
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发表时间：
2011-12-15
影响因子：
2.9
通讯作者：
Zhu, Guoping
中科院分区：
文献类型：
--
作者：
Xu, Lei;Lv, Jun;Zhu, Guoping
在酸性条件下，在一定浓度的胍存在下，在苯酚提取过程中，发现核酸分配到苯酚相中。胍浓度依赖的核酸分配模式类似于由胍介导的核酸吸附/分配到二氧化硅上的模式，这意味着苯酚和二氧化硅通过类似的机制与核酸相互作用。观察到竞争效应，其中已经分配到苯酚相或二氧化硅固相上的核酸可以通过钾以分子量-盐浓度依赖性方式回收到水相中（较高分子量的核酸需要较高浓度的钾来回收，反之亦然）。基于这些发现，开发了从大肠杆菌中分离总RNA的方法。通过控制在苯酚提取或二氧化硅吸附之前使用的胍盐和钾盐的浓度，我们可以选择性地回收水相中的总RNA而不是高分子量基因组DNA。通过我们的方法获得的不含基因组DNA的总RNA适用于RT-PCR或其他目的。通过改变所用的盐浓度，所述方法还可以适于分离小RNA或某些分子量范围内的RNA。(C)2011 Elsevier Inc. All rights reserved.
Nucleic acids were found to partition into the phenol phase during phenol extraction in the presence of guanidinium at certain concentrations under acidic conditions. The guanidinium-concentration-dependent nucleic acid partitioning patterns were analogous to those of the nucleic acid adsorption/partitioning onto silica mediated by guanidinium, which implied that phenol and silica interact with nucleic acids through similar mechanisms. A competition effect was observed in which the nucleic acids that had partitioned into the phenol phase or onto the silica solid phase could be recovered to the aqueous phases by potassium in a molecular weight-salt concentration-dependent manner (the higher molecular weight nucleic acids needed higher concentrations of potassium to be recovered, and vice versa). Methods were developed based on these findings to isolate total RNA from Escherichia coli. By controlling the concentrations of guanidinium and potassium salts used before phenol extraction or silica adsorption, we can selectively recover total RNA but not the high molecular weight genomic DNA in the aqueous phases. Genomic DNA-free total RNA obtained by our methods is suitable for RT-PCR or other purposes. The methods can also be adapted to isolate small RNAs or RNA in certain molecular weight ranges by changing the salt concentrations used. (C) 2011 Elsevier Inc. All rights reserved.