ena-STUDY-UNIVERSITY OF WATERL-23-10-2018-16:06:37:899-265
DNA extraction from natural and spiked MX 80 bentonite
Bentonite is being considered as a part of a multibarrier system for safe long-term storage of high-level radioactive waste. Amplicon-based and meta-omic methods offer enormous potential for assessing the presence and abundance of microorganisms and evaluating their potential for contributing metabolic activity within the barrier environment. The extraction of microbial DNA from bentonite clay is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Successful desorption of nucleic acids from clay using blocking agents has been demonstrated previously but evaluating quantitative recovery of nucleic acids at varying starting concentrations and the subsequent impact of those blocking agents on microbial community profiles have not been characterized well. Using high-throughput sequencing, we assessed DNA extraction efficiency from natural and spiked MX 80 bentonite in the presence of casein and phosphate as blocking agents. We demonstrate improved DNA recovery by blocking agents at high spiking concentration. At low spiking concentrations we detected a high proportion of contaminant sequences, masking sample-specific signals.
Bentonite is being considered as a part of a multibarrier system for safe long-term storage of high-level radioactive waste. Amplicon-based and meta-omic methods offer enormous potential for assessing the presence and abundance of microorganisms and evaluating their potential for contributing metabolic activity within the barrier environment. The extraction of microbial DNA from bentonite clay is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Successful desorption of nucleic acids from clay using blocking agents has been demonstrated previously but evaluating quantitative recovery of nucleic acids at varying starting concentrations and the subsequent impact of those blocking agents on microbial community profiles have not been characterized well. Using high-throughput sequencing, we assessed DNA extraction efficiency from natural and spiked MX 80 bentonite in the presence of casein and phosphate as blocking agents. We demonstrate improved DNA recovery by blocking agents at high spiking concentration. At low spiking concentrations we detected a high proportion of contaminant sequences, masking sample-specific signals.