High-Yield and Phylogenetically Robust Methods of DNA Recovery for Analysis of Microbial Biofilms Adherent to Plant Biomass in the Herbivore Gut
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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.
KeywordsMicrobial Biomass Plant Biomass Sucrose Density Gradient Fosmid Library Sucrose Density Gradient Centrifugation
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.
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