Purification and characterization of a common soil component which inhibits the polymerase chain reaction
Purification and characterization of a common soil component which inhibits the polymerase chain reaction
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DOI：
10.1139/cjm-46-7-633
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发表时间：
2000-07-01
影响因子：
2.8
通讯作者：
Blackwell, B
中科院分区：
文献类型：
--
作者：
Watson, RJ;Blackwell, B
从土壤中制备的DNA通常含有聚合酶链反应（PCR）的棕色抑制剂，这限制了该技术用于微生物特异性检测的灵敏度。为了定位抑制剂，测试土壤组分对PCR反应的抑制作用。一个高度的抑制活性，足以占抑制通常表现出的土壤DNA，被发现是紧密相关的土壤微生物馏分。在细胞破碎后，抑制物质变得可溶，并且不能通过标准纯化程序与DNA分离。一种方法，通过该方法可以选择性地从微生物级分中溶解大部分的抑制性物质，而不破坏细胞，使用不同EDTA浓度的缓冲液连续洗涤。该技术用于分离具有表明其是从土壤中纯化的污染DNA的主要PCR抑制剂的特征的物质。它被发现是一种高分子量的有机水溶性化合物，存在于不同地点的各种土壤中。它被发现是显着不同的，从腐殖酸和富里酸的溶解性能，并在其FT-IR和NMR光谱。它与蛋白质形成复合物，并可通过与Taq DNA聚合酶的相互作用抑制PCR反应。
DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.