Optimization of DNA extraction for quantitative marine bacterioplankton community analysis

The coupling between the presence of predominant bacterial species and particular biogeochemical processes is of primary interest in current microbial ecology. Molecular methods such as microarrays and real-time polymerase chain reaction (PCR) may be used to estimate presence or expression of different genes (e.g., 16S rDNA and nifH) in environmental samples; however, to be quantitative, these methods require a reproducible and efficient DNA extraction protocol. Using picogreen DNA quantification, real-time PCR, and an internal DNA standard, we step-wise examined and optimized a protocol for DNA extraction from centrifuged or filtered seawater samples (vol. 2 to 300 mL). Sample volume had a pronounced effect on DNA extraction efficiency showing that comparison of samples with different volumes is problematic. The duration of enzyme treatment (lysozyme and proteinase K) significantly influenced the DNA extraction efficiency. Dissolved DNA contributed significantly to total DNA when extracted from small filtering volumes (<10 mL). Addition of a coprecipitant (yeast tRNA) improved the precipitation of low-concentration DNA from 13% to 89%. When tested on various seawater samples, as well as on isolates, the optimized extraction protocol was found to be highly reproducible with an average extraction efficiency for seawater samples of 92% and for isolates of 96%. The quantitative outcome and the high extraction efficiency of the presented extraction protocol will be of value to future studies based on DNA extracted from seawater samples. *Corresponding author. E-mail address: [email protected] Acknowledgments We thank Svend Binnerup, National Environmental Research Institute, Denmark, for providing the bacterial isolates, and Karin Holmfeldt and Henrik Hallberg for their assistance in filtering experiments. This work was supported by the Swedish science councils VR (621-2001-2814) and FORMAS (2002-1300) and by the EC through contract EVK3-CT-2002-00078 “BASICS” to ÅH. Limnol. Oceanogr.: Methods 2, 2004, 365–373 © 2004, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS