Preparation and analysis of environmental DNA: optimisation of techniques for phylogenetic analysis of ATAD sludge

peer-reviewedNon-culture based analysis of microbial populations via phylogenetic analysis is becoming an increasingly important tool in the determination of microbial communities and community flux in a large number of environmental niches. The results obtained are however only as good as the preparative techniques used to isolate the original templates and the PCR processing techniques used to amplify these templates. In thermal niches such as autothermal thermophilic aerobic digestion systems (ATAD) there are many features that limit the optimal recovery of microbial diversity. Such features include lysis and release of products from the microbial population such as nucleases and proteases which effect both template recovery and polymerase functionality, inhibitory substances from the environmental source and even the nature of the primers used to amplify the resultant templates. In optimisation of the extraction and amplification of environmental DNA from an ATAD system we have identified a number of issues that affect the recovery of diversity which may have widespread applicability to the recovery of diversity by molecular techniques from other environmental niches. Methods of optimisation and analysis of diversity recovery will be discussed.HE