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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.
 [1] ;  [1] ;  [1] ;  [2] ;  [1]
  1. ORNL
  2. University of Tennessee, Knoxville (UTK)
Publication Date:
OSTI Identifier:
DOE Contract Number:
Resource Type:
Journal Article
Resource Relation:
Journal Name: PLoS ONE; Journal Volume: 9; Journal Issue: 7
Research Org:
Oak Ridge National Laboratory (ORNL)
Sponsoring Org:
SC USDOE - Office of Science (SC)
Country of Publication:
United States
mercury; mercury SFA; mercury methylation; ENIGMA