An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts.
We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants </=100 000 Da. Recovery of DNA using this method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.
Kallmeyer
Jens
J
Graduate School of Oceanography, University of Rhode Island, Narragansett, RI, USA. jens.kallmeyer@geo.uni-potsdam.de
Smith
David C
DC
D004247:DNA / Q000302:isolation & purification*; D019641:DNA, Archaeal / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D019015:Geologic Sediments / Q000032:analysis*; D006812:Humic Substances / Q000032:analysis*; D016133:Polymerase Chain Reaction
D019641:DNA, Archaeal; D004269:DNA, Bacterial; D006812:Humic Substances; D004247:DNA