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

✍ Scribed by S.K. Juniper; M.-A. Cambon; F. Lesongeur; G. Barbier


Book ID
104156938
Publisher
Elsevier Science
Year
2001
Tongue
English
Weight
842 KB
Volume
174
Category
Article
ISSN
0025-3227

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✦ Synopsis


Molecular biology offers many new tools for the characterisation of mixed communities of microorganisms. Approaches that require the extraction and puri®cation 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 puri®cation 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 378C), 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 puri®cation procedures (phenol/chloroform extraction followed by silicabased 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 ampli®cation and cloning of SSU (small subunit) rRNA genes. A detailed extraction and puri®cation protocol is presented. These procedures, although time-consuming, may be applicable to other sediment types where microbial DNA is particularly dif®cult to extract or purify.


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