Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China.
World J Microbiol Biotechnol. 2013 May;29(5):815-23. doi: 10.1007/s11274-012-1236-6. Epub 2012 Dec 14.
An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze-thaw-SDS-Protein K) and FTSPP (Freeze-thaw-SDS-Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL(-1) BSA. However, the FTSPP extraction method with DNA purification by a Wizard(®) kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.
对于通过核酸技术分析遗传多样性，特别是分析复杂环境样本中的基因，需要采用无偏的 DNA 提取方案。在本研究中，我们比较了三种手动提取方法和两种常用的商用试剂盒，这些方法均采用两种 DNA 纯化策略，比较的依据是细胞裂解效率、DNA 和腐殖酸得率、PCR 扩增和变性梯度凝胶电泳（DGGE）分析。结果表明，尽管两种商用试剂盒的细胞裂解效率较高，但纯化的 DNA 产量仅为两种手动方法（FTSP，冻融- SDS-蛋白酶 K 和 FTSPP，冻融- SDS-蛋白酶 K-聚乙烯基吡咯烷酮）的三分之一。所有五种方法获得的纯化 DNA 均足够纯净，可在存在 1μgμL(-1)BSA 的情况下成功进行 PCR 和实时 PCR 扩增。然而，采用 Wizard(®)试剂盒进行 DNA 纯化的 FTSPP 提取方法在 DGGE 图谱中产生了最大数量的 16S rRNA 基因拷贝和核糖体型或条带，这表明其优于其他四种方法。该优化的 DNA 提取和纯化方法的开发可为进一步研究堆肥的分子分析提供有价值的工具。
