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Abstract
Volume 6, Issue 2, April Issue - 2018, Pages:315-323 |
| Authors: P. Verma, V. Vasudevan, B. K. Kashyap, T. I. Samsudeen, M. K. Meghvansi, D. V. Kamboj, L. Singh |
| Abstract: DNA extraction from anaerobic biodigester slurry is a critical step in all phylogenetic and metagenomic approaches to characterize highly diverse biodigester ecosystem, but little is known about the efficiency of different extraction procedures and their impact on subsequent analyses of microbial communities. The assessment of performance differences, therefore, is a concrete step towards the determination of optimal DNA extraction method for biodigester slurry, which can provide a more reliable comparison of the meta-analysis results obtained in different conditions. Here, we report a highly efficient direct lysis genomic DNA extraction method (Glass milk method) from the slurry of an anaerobic biodigester. This method was compared with five commercially available DNA extraction kits and two different manual methods with regard to DNA extraction, purification efficiencies and representation of archaeal diversity through PCR-Denaturing Gradient Gel Electrophoresis (DGGE). Results revealed that commercial kits yielded significantly lower DNA concentrations than manual methods. Moreover, interpretations of bacterial community structure based on analysis of PCR-DGGE banding pattern of 16S rRNA gene, Shannon-Wiener and Simpson’s indices of diversity and multidimensional scaling suggested that manual methods of DNA isolation revealed far greater archaeal diversity as compared to the commercial kits. Further, in real-time PCR analysis, a methanogens-specific 16S rRNA gene concentration was observed more in manual methods than in commercial kits. The glass milk DNA extraction method proposed here is very useful in pursuing phylogenetic and metagenomic approaches to characterize the highly complex anaerobic biodigester ecosystem with greater reliability and precision. |
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