Extraction and purification of DNA from organic rich subsurface sediments (ODP Leg 169S)

Molecular biology offers many new tools for the characterisation of mixed communities of microorganisms. Approaches that require the extraction and purification of bulk community DNA from sediments and soils must contend with contaminants such as humic acids and heavy metals that can interfere with subsequent genetic analysis. This paper reports on the adaptation of DNA extraction and purification techniques to samples of organic rich sediments collected during the Ocean Drilling Program Leg 169S in Saanich Inlet, British Columbia. In an extraction time series, DNA yield increased up to 48 h (at 37 degreesC), after which there were negligible increases in yield and signs of degradation. Resulting extracts, rich in humic substances, blocked the DNA polymerase enzyme even at high dilution. Standard purification procedures (phenol/chloroform extraction followed by silica-based DNA binding or agarose gel electrophoresis) proved ineffective in removing PCR inhibitors. The inhibitory effect was eliminated by cesium chloride density gradient centrifugation with eukaryote DNA added as a carrier, permitting amplification and cloning of SSU (small subunit) rRNA genes. A detailed extraction and purification protocol is presented. These procedures, although time-consuming, may be applicable to other sediment types where microbial DNA is particularly difficult to extract or purify. (C) 2001 Elsevier Science B.V. All rights reserved.