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

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.
Authors:
 [1] ;  [1] ;  [2] ;  [1] ;  [2] ;  [3] ;  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Div.
  2. Univ. of Tennessee, Knoxville, TN (United States). Dept. of Microbiology
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Environmental Sciences Div.
Publication Date:
OSTI Identifier:
1207041
Grant/Contract Number:
AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 9; Journal Issue: 7; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; mercury; mercury SFA; mercury methylation; ENIGMA