Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

Hurt, Jr., Richard A.; Robeson II, Michael S.; Shakya, Migun; Moberly, James G.; Vishnivetskaya, Tatiana; Gu, Baohua; Elias, Dwayne A.

doi:10.1371/journal.pone.0102826

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

Journal Article · Mon Jul 14 00:00:00 EDT 2014 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0102826· OSTI ID:1207041

Hurt, Jr., Richard A. ^[1]; Robeson II, Michael S. ^[1]; Shakya, Migun ^[2]; Moberly, James G. ^[1]; Vishnivetskaya, Tatiana ^[2]; Gu, Baohua ^[3]; Elias, Dwayne A. ^[1]

Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Div.
Univ. of Tennessee, Knoxville, TN (United States). Dept. of Microbiology
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Environmental Sciences Div.

Despite more than three decades of progress, efficient nucleic acid extraction from microbial communities has remained difficult, particularly from clay environments. Lysis with concentrated guanidine followed by concentrated sodium phosphate extraction supported DNA and RNA recovery from high iron, low humus content clay. Alterating the extraction pH or using other ionic solutions (Na₂SO₄ and NH₄H₂PO₄) yielded no detectable nucleic acid. DNA recovered using a lysis solution with 500 mM phosphate buffer (PB) followed by a 1 M PB wash was 15.22±2.33 g DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 g DNA/g clay with the Powerlyzer soil DNA system (MoBio). Increasing [PB] in the lysis reagent coincided with increasing DNA fragment length. Rarefaction plots based on16S rRNA (V1/V3 region) pyrosequencing libraries from A-horizon and clay soils showed an ~80% and ~400% larger accessed diversity compared to a previous grinding protocol or the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more bacterial species recovered using this system. Additionally, some OTU's having more than 100 sequences in these libraries were absent in samples extracted using the PowerLyzer reagents or the previous lysis method.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1207041

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 7 Vol. 9; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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