Yang Zhao-Hui, Xiao Yong, Zeng Guang-Ming, Liu Yun-Guo, Deng Jiu-Hua
College of Environmental Science and Engineering, Hunan University, Changsha 410082, China.
Huan Jing Ke Xue. 2006 Aug;27(8):1613-7.
Molecular ecology provides new techniques for studying compost microbes, and the DNA extraction is the basis of molecular techniques. Because of the contamination of humic acids, it turns to be more difficult for compost microbial DNA extraction. Three different approaches, named as lysozyme lysis, ultrasonic lysis and proteinase K lysis with CTAB, were used to extract the total DNA from compost. The detection performed on a nucleic acids and protein analyzer showed that all the three approaches produced high DNA yields. The agarose gel electrophoresis showed that the DNA fragments extracted from compost had a length of about 23 kb. A eubacterial 16S rRNA gene targeted primer pair (27F and 1 495R) was used for PCR amplification, and all the samples got almost the full length 16S rDNA sequence (about 1.5 kb). After digested by restriction endonucleases (Hae Ill and Alu I), the restriction map showed relatively identical microbial diversity in the DNA, which was extracted by the three different approaches. All the compost microbial DNA extracted by the three different approaches could be used for molecular ecological study, and researchers should choose the right approach for extracting microbial DNA from compost based on the facts.
分子生态学为研究堆肥微生物提供了新的技术,而DNA提取是分子技术的基础。由于腐殖酸的污染,堆肥微生物DNA的提取变得更加困难。采用三种不同的方法,即溶菌酶裂解、超声裂解和CTAB蛋白酶K裂解,从堆肥中提取总DNA。在核酸和蛋白质分析仪上进行的检测表明,这三种方法都产生了高产量的DNA。琼脂糖凝胶电泳显示,从堆肥中提取的DNA片段长度约为23 kb。使用一对针对真细菌16S rRNA基因的引物(27F和1495R)进行PCR扩增,所有样品几乎都获得了全长16S rDNA序列(约1.5 kb)。经限制性内切酶(Hae III和Alu I)消化后,限制性图谱显示三种不同方法提取的DNA中微生物多样性相对相同。三种不同方法提取的所有堆肥微生物DNA均可用于分子生态学研究,研究人员应根据实际情况选择合适的方法从堆肥中提取微生物DNA。