Improved purification and PCR amplification of DNA from environmental samples
Improved purification and PCR amplification of DNA from environmental samples
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DOI：
10.1111/j.1574-6968.2007.00764.x
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发表时间：
2007-07-01
影响因子：
2.1
通讯作者：
Fuentes, Cilia L.
中科院分区：
文献类型：
--
作者：
Arbeli, Ziv;Fuentes, Cilia L.
从环境样品（土壤，堆肥，河流沉积物）提取的DNA的纯化和PCR扩增程序进行了改进，通过引入三个修改：沉淀的DNA与5%的聚乙二醇8000（PEG）和0.6 M NaCl;过滤与Sepharose 4 B-聚乙烯吡咯烷酮（PVPP）旋转柱;和添加脱脂牛奶（0.3% w/v）的PCR反应溶液。用5%PEG沉淀后的腐殖物质浓度分别比用7.5%PEG、10%PEG和异丙醇沉淀低2.57倍、5.3倍和78.9倍。PEG沉淀后，Sepharose，PVPP和组合（Sepharose-PVPP）柱去除92.3%，89.5%和98%，分别剩余的腐殖物质。上述每种修饰都改善了16 S rRNA基因的PCR扩增。所提出的方案提取的DNA比商业试剂盒提取的DNA更干净。然而，DNA纯化的改进并没有提高阿特拉津降解基因atzA的检测限。
Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.