Abstract
Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery. DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes, and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library demonstrated that the recovered HMW DNA is suitable for metagenomic library construction.
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Acknowledgements
This research was partially funded by the Knowledge Innovation Program of the Chinese Academy of Sciences (KZCX2-YW-216, KZCX2-YW-211), National Supportive Plan Project of Science and Technology (2006BAB19B02), and the Research Foundation of Science and Technology Plan Project in Guangdong Province (2008A030203004). We also thank the financial support of the Hundred Talents Program of Chinese Academy of Sciences.
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Yongchang Ouyang and Shikun Dai contributed equally to this work.
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Ouyang, Y., Dai, S., Xie, L. et al. Isolation of High Molecular Weight DNA from Marine Sponge Bacteria for BAC Library Construction. Mar Biotechnol 12, 318–325 (2010). https://doi.org/10.1007/s10126-009-9223-0
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DOI: https://doi.org/10.1007/s10126-009-9223-0