Purification of total DNA extracted from activated sludge  

作　　者：SHAN Guobin JIN Wenbiao Edward KHLAM XING Xinhui 

机构地区：[1]Baolimei Chemical Engineering Co., Ltd., Dongguan 523581, China. [2]Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China [3]Department of Urban and Civil Engineering, Harbin Institute of Technology Shenzhen Graduate School, Shenzhen 518055, China

出　　处：《Journal of Environmental Sciences》2008年第1期80-87,共8页环境科学学报（英文版）

基　　金：This work was supported by the China Postdoctoral Science Foundation(No.20060390060).

摘　　要：Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments （lysozyme, sodium dodecyl sulfate （SDS）, sonication, mechanical mill and thermal shock） on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg （total DNA）/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis （denatured gradient gel electrophoresis） to be possible.Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments （lysozyme, sodium dodecyl sulfate （SDS）, sonication, mechanical mill and thermal shock） on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg （total DNA）/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis （denatured gradient gel electrophoresis） to be possible.

关 键 词：16S rDNA activated sludge PCR lysis treatment DNA purification wasterwater biotreatment 

分 类 号：X703[环境科学与工程—环境工程]