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

Author(s): Juniper, S. K.; Cambon, M. A.; Lesongeur, F.; Barbier, G.
Author Affiliation(s): Primary:
IFREMER, Centre de Brest, Plouzane, France
Other:
University of Southampton, United Kingdom
Volume Title: Late Quaternary sedimentation in Saanich Inlet, British Columbia, Canada; Ocean Drilling Program Leg 169S
Volume Author(s): Bornhold, Brian D., editor; Kemp, Alan E. S.
Source: Late Quaternary sedimentation in Saanich Inlet, British Columbia, Canada; Ocean Drilling Program Leg 169S, edited by Brian D. Bornhold and Alan E. S. Kemp. Marine Geology, 174(1-4), p.241-247. Publisher: Elsevier, Amsterdam, Netherlands. ISSN: 0025-3227 CODEN: MAGEA6
Note: In English. 24 refs.; illus.
Summary: 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 48h (at 37°C), 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. Abstract Copyright (2001) Elsevier, B.V.
Year of Publication: 2001
Research Program: ODP Ocean Drilling Program
Key Words: 02 Geochemistry; Biochemistry; British Columbia; Canada; Chemical composition; DNA; Leg 169S; Marine sedimentation; Marine sediments; ODP Site 1033; Ocean Drilling Program; Organic compounds; Saanich Inlet; Sedimentation; Sediments; Western Canada
Coordinates: N483000 N483000 W1231500 W1231500
Record ID: 2001043091
Copyright Information: GeoRef, Copyright 2019 American Geosciences Institute. Reference includes data from CAPCAS, Elsevier Scientific Publishers, Amsterdam, Netherlands