| 苟敏,曲媛媛,周集体,曹湘禹,许炳雯,王腾.活性污泥宏基因组DNA提取方法优化[J].,2012,(6):803-808 |
| 活性污泥宏基因组DNA提取方法优化 |
| Optimization of metagenomic DNA extraction from activated sludge |
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| DOI：10.7511/dllgxb201206005 |
| 中文关键词: 宏基因组DNA 活性污泥 单因素法 表面响应法 |
| 英文关键词: metagenomic DNA activated sludge single-factor method response surface methodology |
| 基金项目:国家自然科学基金资助项目(51078054,20923006). |
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| 中文摘要: |
| 宏基因组DNA的成功获取是宏基因组学技术顺利开展的关键．利用溶菌酶-SDS-蛋白酶K法(LSK法)提取活性污泥宏基因组DNA,结合单因素法与表面响应法对提取过程的4个关键步骤进行条件优化,即预处理、细胞裂解、蛋白去除及DNA沉淀过程．确定影响DNA产量的重要因素:预处理缓冲溶液与DNA沉淀剂类型、CTAB质量分数、溶菌酶浓度、37 ℃水浴时间及SDS质量分数．确定LSK法提取宏基因组DNA的最佳条件:TENC预处理活性污泥；1.5% CTAB；0.5 mg/mL溶菌酶,37 ℃水浴1.4 h；2% SDS,200 μg/mL蛋白酶,55 ℃水浴2.5 h；异丙醇沉淀DNA 40 min．在最佳条件下,活性污泥宏基因组DNA的最大产量为每g污泥170 μg．本研究可为环境样品中高质量宏基因组DNA的提取提供有价值的参考． |
| 英文摘要: |
| The extraction of metagenomic DNA is the key step for metagenomic technology. Lysozyme-SDS-proteinase K method (LSK method) is used to extract metagenomic DNA from activated sludge. Four steps of LSK method, such as pretreatment, cell lysis, protein removal and DNA precipitation, are optimized using single-factor method and response surface methodology (RSM). And several key factors for DNA extraction are identified, including the type of buffer for pretreatment and reagent for DNA precipitation, CTAB mass fraction, lysozyme concentration, SDS mass fraction and the time of water bath at 37 ℃. The optimal conditions for DNA extraction using LSK method are as follows: TENC as pretreatment buffer, 1.5% of CTAB, 0.5 mg/mL of lysozyme, water bath at 37 ℃ for 1.4 h, 2% of SDS, 200 μg/mL of proteinase, water bath at 55 ℃ for 2.5 h, DNA precipitation for 40 min with isopropanol. The highest DNA yield is obtained with 170 μg per gram sludge under the optimal conditions. This study will provide valuable reference for extraction of high-quality metagenomic DNA from environmental samples. |
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