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Microbial Ecology

, Volume 61, Issue 2, pp 448–454 | Cite as

High-Yield and Phylogenetically Robust Methods of DNA Recovery for Analysis of Microbial Biofilms Adherent to Plant Biomass in the Herbivore Gut

  • Carly P. Rosewarne
  • Phillip B. Pope
  • Stuart E. Denman
  • Christopher S. McSweeney
  • Paraic O’Cuiv
  • Mark MorrisonEmail author
Methods

Abstract

Recent studies have shown the microbial biofilms adherent to plant biomass in the gastrointestinal tracts of humans and other herbivores are quite different to planktonic populations. If these biofilm communities are to be properly characterized by metagenomics methods, then the microbial desorption methods used must ensure the phylogenetic diversity and genetic potential recovered is biologically valid. To that end, we describe here two different methods for desorbing microbes tightly adherent to plant biomass; and used PCR-DGGE analyses of the Bacteria and Archaea rrs genes to show both these desorption methods were effective in recovering the adherent microbial biofilm with no apparent biases in microbe recovery. We also present a derivation of the “repeated bead beating and column (RBB+C) purification” method of DNA extraction that results in the recovery of high molecular weight DNA. These DNA samples can be fragmented and size fractionated by sucrose density gradient centrifugation, bypassing the use of gel-plug lysis and pulsed-field gel electrophoresis separation of DNA for metagenomic library constructions.

Keywords

Microbial Biomass Plant Biomass Sucrose Density Gradient Fosmid Library Sucrose Density Gradient Centrifugation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We thank Rafat Al-Jassim (Department of Animal Studies, University of Queensland, Gatton) for the provision of freshly collected rumen digesta. We also thank Seungha Kang for helpful suggestions during the preparation of this manuscript. This research has been partially supported with funds provided by a CSIRO Australia OCE postdoctoral fellowship scheme (to PBP) as well as funds provided by Meat and Livestock Australia project number B.CCH.1005, as part of the Reducing Emissions in Livestock Research Program.

Supplementary material

248_2010_9745_MOESM1_ESM.doc (38 kb)
Figure S1 Agarose gel electrophoresis of PCR products generated from chloroplast DNA. Approximately 100 ng of DNA was used as the template in each lane. M 100 bp DNA ladder (New England Biolabs); 1 direct RBB+C DNA extraction from ingested plant biomass; 2 RBB+C DNA extraction from cells desorbed with repeated vortexing method; 3 RBB+C DNA extraction from cells desorbed with Larue et al. [15] method; 4 HMW DNA extraction from cells desorbed with repeated vortexing method; 5 HMW DNA extractions from cells desorbed with Larue et al. [15] method; 6 positive control; 7 no template control (DOC 156 kb)
248_2010_9745_MOESM2_ESM.doc (75 kb)
Figure S2 Pulsed-field gel electrophoresis of sucrose density gradient fractions F19-27 isolated from desorbed microbial biofilms with the NML method. M Low-range PFG marker (New England Biolabs; bands at 23.1 and 48 kb as indicated); C fosmid control DNA (36 kb) (DOC 75 kb)
248_2010_9745_MOESM3_ESM.doc (86 kb)
Figure S3 Pulsed-field gel electrophoresis showing examples of purified blunt-end DNA from selected sucrose density gradient fractions. M indicates the λ HinDIII marker from which the sizes of selected fragments are indicated in kbp. C indicates the fosmid control DNA which is 36 kb in size (DOC 86 kb)

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Carly P. Rosewarne
    • 1
  • Phillip B. Pope
    • 1
  • Stuart E. Denman
    • 1
  • Christopher S. McSweeney
    • 1
  • Paraic O’Cuiv
    • 1
  • Mark Morrison
    • 1
    • 2
    Email author
  1. 1.Commonwealth Scientific and Industrial Research Organisation (CSIRO), Division of Livestock Industries, Queensland Bioscience PrecinctSt. LuciaAustralia
  2. 2.Department of Animal SciencesThe Ohio State UniversityColumbusUSA

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