Lim Hyun Jeong, Choi Jung-Hyun, Son Ahjeong
Department of Environmental Science and Engineering, Ewha Womans University, Seoul, Korea.
Environ Health Toxicol. 2017 Aug 8;32:e2017013. doi: 10.5620/eht.e2017013. eCollection 2017.
Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content.
土壤样品的复杂性和异质性常常意味着在用于聚合酶链反应（PCR）分析的传统DNA提取过程中需要加入纯化步骤。不幸的是，这些纯化步骤既耗时又费力。因此，针对含有不同量PCR抑制剂的各种环境土壤样品，对DNA纯化的必要性进行了重新审视和研究。珠磨法和离心法被用作DNA提取的基线（不进行纯化）方法。将其性能与传统DNA提取试剂盒（进行纯化）的性能进行了比较。利用环境土壤样品确定了DNA纯化的必要标准。在使用0.1毫米玻璃珠、3000转/分钟的裂解条件下处理3分钟、离心10分钟以及1:10的稀释比例时，基线方法在接种细胞的砂质样品上的表现优于传统DNA提取方法。对PCR抑制剂（即腐殖酸、黏土和镁[Mg]）的进一步研究表明，腐殖酸含量低于10μg/g且黏土含量低于70%的砂质样品可能不需要纯化。有趣的是，由于镁离子与DNA片段的络合相互作用，镁离子的抑制模式与其他抑制剂不同。得出的结论是，不进行纯化的DNA提取方法适用于腐殖酸含量低于10μg/g、黏土含量低于70%且镁离子含量低于0.01%的土壤样品。
