Validating DNA Extraction Protocols for Bentonite Clay

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data re...

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Autores principales:	Katja Engel, Sara Coyotzi, Melody A. Vachon, Jennifer R. McKelvie, Josh D. Neufeld
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Wyoming MX-80 bentonite clay bacteria DNA extraction casein Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/04de3d83d21a49aca7f67fc953966b44
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spelling	oai:doaj.org-article:04de3d83d21a49aca7f67fc953966b442021-11-15T15:27:32ZValidating DNA Extraction Protocols for Bentonite Clay10.1128/mSphere.00334-192379-5042https://doaj.org/article/04de3d83d21a49aca7f67fc953966b442019-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00334-19https://doaj.org/toc/2379-5042ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.Katja EngelSara CoyotziMelody A. VachonJennifer R. McKelvieJosh D. NeufeldAmerican Society for MicrobiologyarticleWyoming MX-80bentoniteclaybacteriaDNA extractioncaseinMicrobiologyQR1-502ENmSphere, Vol 4, Iss 5 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Wyoming MX-80 bentonite clay bacteria DNA extraction casein Microbiology QR1-502
spellingShingle	Wyoming MX-80 bentonite clay bacteria DNA extraction casein Microbiology QR1-502 Katja Engel Sara Coyotzi Melody A. Vachon Jennifer R. McKelvie Josh D. Neufeld Validating DNA Extraction Protocols for Bentonite Clay
description	ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.
format	article
author	Katja Engel Sara Coyotzi Melody A. Vachon Jennifer R. McKelvie Josh D. Neufeld
author_facet	Katja Engel Sara Coyotzi Melody A. Vachon Jennifer R. McKelvie Josh D. Neufeld
author_sort	Katja Engel
title	Validating DNA Extraction Protocols for Bentonite Clay
title_short	Validating DNA Extraction Protocols for Bentonite Clay
title_full	Validating DNA Extraction Protocols for Bentonite Clay
title_fullStr	Validating DNA Extraction Protocols for Bentonite Clay
title_full_unstemmed	Validating DNA Extraction Protocols for Bentonite Clay
title_sort	validating dna extraction protocols for bentonite clay
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/04de3d83d21a49aca7f67fc953966b44
work_keys_str_mv	AT katjaengel validatingdnaextractionprotocolsforbentoniteclay AT saracoyotzi validatingdnaextractionprotocolsforbentoniteclay AT melodyavachon validatingdnaextractionprotocolsforbentoniteclay AT jenniferrmckelvie validatingdnaextractionprotocolsforbentoniteclay AT joshdneufeld validatingdnaextractionprotocolsforbentoniteclay
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Validating DNA Extraction Protocols for Bentonite Clay

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