Yang Zh H, Xiao Y, Zeng G M, Xu Zh Y, Liu Y Sh
College of Environmental Science and Engineering, Hunan University, Changsha, Hunan Province, China.
Appl Microbiol Biotechnol. 2007 Mar;74(4):918-25. doi: 10.1007/s00253-006-0704-z. Epub 2006 Nov 18.
The differences on DNA yield and purity of three different DNA extraction protocols were compared with regard to the use for PCR and other molecular analyses. Total DNA was extracted from compost by the three protocols, and then was purified by spin-bind cartridges after being precipitated by PEG8000. The detection performed on a nucleic acid and protein analyzer showed that all three methods produced high DNA yields. The agarose gel electrophoresis showed that the fragments of crude and purified DNA had a length of about 23 kb. A eubacterial 16S rRNA gene-targeted primer pair was used for PCR amplification, and full length 16S rDNAs were amplified from all the purified DNA samples. After being digested by restriction endonucleases, the restriction map of amplified rDNA showed identical genetic diversity. The products of PCR using primer pair GC341F and 907R were also used for denaturing gradient gel electrophoresis analysis. The results indicated that high-quality DNA was extracted from compost by the three protocols, and each of the protocols is adapted to extract microbial genome DNA from compost expediently and cheaply.
比较了三种不同DNA提取方案在用于PCR和其他分子分析时DNA产量和纯度的差异。采用这三种方案从堆肥中提取总DNA，然后在经PEG8000沉淀后通过离心柱进行纯化。在核酸和蛋白质分析仪上进行的检测表明，所有三种方法都产生了高DNA产量。琼脂糖凝胶电泳显示，粗制和纯化后的DNA片段长度约为23 kb。使用一对靶向真细菌16S rRNA基因的引物进行PCR扩增，从所有纯化的DNA样品中均扩增出全长16S rDNA。经限制性内切酶消化后，扩增rDNA的限制性图谱显示出相同的遗传多样性。使用引物对GC341F和907R进行PCR的产物也用于变性梯度凝胶电泳分析。结果表明，这三种方案均能从堆肥中提取出高质量的DNA，且每种方案都适合于方便、廉价地从堆肥中提取微生物基因组DNA。
