RAPID AND SIMPLE METHOD FOR PURIFICATION OF NUCLEIC-ACIDS
RAPID AND SIMPLE METHOD FOR PURIFICATION OF NUCLEIC-ACIDS
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DOI：
10.1128/jcm.28.3.495-503.1990
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发表时间：
1990-03-01
影响因子：
9.4
通讯作者：
VANDERNOORDAA, J
中科院分区：
文献类型：
--
作者：
BOOM, R;SOL, CJA;VANDERNOORDAA, J
我们已经开发了一种简单、快速和可靠的方案，用于小规模纯化DNA和RNA，人血清和尿液。该方法基于离液剂硫氰酸胍的裂解和裂解以及核酸酶失活特性，以及在该试剂存在下二氧化硅颗粒或硅藻的核酸结合特性。通过使用大小分级的二氧化硅颗粒，核酸（共价闭合环状，松弛环状，松弛环状和线性双链DNA;单链DNA;和rRNA）可以在不到1小时内从12个不同的标本中纯化，并在初始反应容器中回收。纯化的DNA（虽然明显剪切）是限制性内切酶和DNA连接酶的良好底物，并以高收率（通常超过50%）从皮克到微克水平回收。回收的共纯化rRNA几乎未降解。用大小分级的二氧化硅颗粒代替硅藻（单细胞藻类的细胞壁），可以从富含细胞的来源中纯化出微克量的基因组DNA、质粒DNA和rRNA，例如致病性革兰氏阴性细菌。在本文中，我们展示了具有代表性的实验，说明了该程序的一些特点，这些特点可能在临床微生物学中具有广泛的应用。
We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.