Improvements on environmental DNA extraction and purification procedures for matagenomic analysis

• Published in: Journal of Central South University Vol. 19; no. 11; pp. 3055 - 3063
• Main Authors: Xie, Jian-ping, Wu, Li-you, van Nostrand, J. D., He, Zhi-li, Lü, Zhen-mei, Yu, Hao, Xiong, Jin-bo, Liu, Xin-xing, Zhou, Ji-zhong
• Format: Journal Article
• Language: English
• Published: Heidelberg Central South University 01.11.2012
• Subjects: Engineering; Metallic Materials; pyrosequencing; DNA extraction; metagenomic analysis; DNA purification; GeoChip
• ISSN: 2095-2899; 2227-5223
• DOI: 10.1007/s11771-012-1378-6

Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers (PIPES and Tris-HCl) and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer (pH 6.5) is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HCl buffer (pH 8.0). Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed: one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.