【摘要】 活性污泥样品经液氮速冻、沸水浴融化、溶菌酶处理和 SDS裂解后 ,99%以上细胞裂解。所提取的 DNA经琼脂糖凝胶电泳检测和荧光法浓度测定 ,其片断大小在 2 0 kb左右 ,产量可达 1 .75 6± 0 .1 mg/g MLSS。样品 ABS2 6 0 nm/ABS2 80 nm的比值为 1 .96± 0 .2。以提取的总 DNA为模板 ,进行细菌核糖体小亚基 1 6Sr DNA基因 V3区和多组分苯酚羟化酶大亚基基因 (Lm PHs)的 PCR扩增 ,均获得成功 ,为活性污泥中微生物群落的分子生态学研究提供了一种简便、可靠的 DNA提取方法。更多还原

【Abstract】 A rapid and less instrument\|dependent protocol for direct extraction of DNA from activated sludge sample was developed. To optimize the cell lysis step, four cell lysis treatments (Freeze\|thaw, lysozyme, SDS , and thermal shock +lysozyme + SDS) were used to disrupt dispersed cells and release DNA. The efficiency of cell lysis was assessed microscopically after each treatment step. The quality and quantity of extracted DNA was determined by using both agarose gel electrophoresis and spectrophotometric measurement. PCR amplification for 16S rDNA and LmPHs (large subunit of phenol hydroxylase) were performed to determine if the DNA was of high enough quality for bacterial community structure and diversity analysis. Results showed that the combination of freeze\|thaw, lysozyme incubationt and SDS lysis was the most efficient one, leading to more than 99% cell disruption. Isolated DNA was over 20kb in size and DNA yield of 1.756±0.1mg·g -1 MLSS was achieved. The ABS 260nm /ABS 280nm of DNA sample was 1 96±0 2,which indicated that the extracted DNA was essentially free from protein contaminants. The successful amplification of V3 region of 16S rDNA, and fragment of the large subunit of the phenol hydroxylase(LmPHs)further demonstrated that the DNA samples were free of inhibitors that interfere with PCR analysis. This protocol has potential for molecular microbial ecology work in activated sludge systems.更多还原