High-yield and phylogenetically robust methods of DNA recovery for analysis of microbial biofilms adherent to plant biomass in the herbivore gut.
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.
Rosewarne
Carly P
CP
Commonwealth Scientific and Industrial Research Organisation, Division of Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, QLD 4067, Australia.
Pope
Phillip B
PB
Denman
Stuart E
SE
McSweeney
Christopher S
CS
O'Cuiv
Paraic
P
Morrison
Mark
M
D000818:Animals; D044822:Biodiversity; D018441:Biofilms; D018533:Biomass; D002417:Cattle / Q000382:microbiology; D019641:DNA, Archaeal / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods; D008297:Male; D010944:Plants / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012417:Rumen / Q000382:microbiology*
D019641:DNA, Archaeal; D004269:DNA, Bacterial