Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, USA.
PLoS One. 2012;7(12):e51863. doi: 10.1371/journal.pone.0051863. Epub 2012 Dec 17.
While most nucleic acids are intracellular, trace amounts of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including micro RNAs, can also be found in peripheral blood. Many studies have suggested the potential utility of these circulating nucleic acids in prenatal diagnosis, early cancer detection, and the diagnosis of infectious diseases. However, DNA circulating in blood is usually present at very low concentrations (ng/ml), and is in the form of relatively small fragments (<1,000 bp), making its isolation challenging.
Here we report an improved method for the isolation of small DNA fragments from serum using selective precipitation by quaternary ammonium compaction agents. A 151 bp fragment of double-stranded DNA from the Escherichia coli bacteriophage lambda served as the model DNA in our experiments. DNA was serially diluted in serum until undetectable by conventional polymerase chain reaction (PCR), before being enriched by compaction precipitation.
Starting with concentrations two to three orders of magnitude lower than the PCR-detectable level (0.01 ng/ml), we were able to enrich the DNA to a detectable level using a novel compaction precipitation protocol. The isolated DNA product after compaction precipitation was largely free of serum contaminants and was suitable for downstream applications.
Using compaction precipitation, we were able to isolate and concentrate small DNA from serum, and increase the sensitivity of detection by more than four orders of magnitude. We were able to recover and detect very low levels (0.01 ng/ml) of a small DNA fragment in serum. In addition to being very sensitive, the method is fast, simple, inexpensive, and avoids the use of toxic chemicals.
虽然大多数核酸都存在于细胞内，但外周血中也能检测到微量脱氧核糖核酸（DNA）和核糖核酸（RNA），包括 microRNAs。这些循环核酸在产前诊断、早期癌症检测和传染病诊断中具有潜在的应用价值，许多研究已经表明了这一点。然而，血液中循环的 DNA 通常浓度很低（ng/ml），而且以相对较小的片段形式存在（<1000bp），因此其分离具有挑战性。
本文报道了一种从血清中分离小 DNA 片段的改良方法，该方法使用季铵盐压实剂选择性沉淀。以大肠杆菌噬菌体 lambda 的双链 DNA 的 151bp 片段作为实验中的模型 DNA。在进行浓缩沉淀之前，先将 DNA 在血清中进行连续稀释，直至经常规聚合酶链反应（PCR）无法检测到。
从比 PCR 可检测水平低两到三个数量级的起始浓度（0.01ng/ml）开始，我们能够使用新的压实沉淀方案将 DNA 浓缩至可检测水平。经压实沉淀后分离得到的 DNA 产物几乎不含血清污染物，适用于下游应用。
使用压实沉淀法，我们能够从血清中分离和浓缩小 DNA，并将检测灵敏度提高四个数量级以上。我们能够从血清中回收和检测到极低水平（0.01ng/ml）的小 DNA 片段。该方法除了非常灵敏之外，还快速、简单、廉价，且避免使用有毒化学品。
