Marine Biotechnology

, Volume 12, Issue 3, pp 318–325

Isolation of High Molecular Weight DNA from Marine Sponge Bacteria for BAC Library Construction

  • Yongchang Ouyang
  • Shikun Dai
  • Lianwu Xie
  • M. S. Ravi Kumar
  • Wei Sun
  • Huimin Sun
  • Danling Tang
  • Xiang Li
Original Article

DOI: 10.1007/s10126-009-9223-0

Cite this article as:
Ouyang, Y., Dai, S., Xie, L. et al. Mar Biotechnol (2010) 12: 318. doi:10.1007/s10126-009-9223-0

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.

Keywords

DNA recovery Marine sponge Bacterial artificial chromosome Metagenomic library 

Supplementary material

10126_2009_9223_MOESM1_ESM.pdf (42 kb)
Supplementary Table 1 (PDF 43 KB)
10126_2009_9223_MOESM2_ESM.pdf (27 kb)
Supplementary Table 2 (PDF 27 KB)
10126_2009_9223_MOESM3_ESM.pdf (18 kb)
Supplementary Table 3 (PDF 18 KB)
10126_2009_9223_MOESM4_ESM.pdf (39 kb)
Supplementary Table 4 (PDF 40 KB)

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Yongchang Ouyang
    • 1
    • 2
  • Shikun Dai
    • 1
    • 2
  • Lianwu Xie
    • 1
    • 2
  • M. S. Ravi Kumar
    • 1
    • 2
    • 3
  • Wei Sun
    • 1
    • 2
  • Huimin Sun
    • 1
    • 2
  • Danling Tang
    • 3
  • Xiang Li
    • 1
    • 4
  1. 1.LMB-CAS, LMMM-GD, South China Sea Institute of OceanologyChinese Academy of SciencesGuangzhouPeople’s Republic of China
  2. 2.Graduate University of the Chinese Academy of SciencesBeijingPeople’s Republic of China
  3. 3.LED, RSMEE, South China Sea Institute of OceanologyChinese Academy of SciencesGuangzhouPeople’s Republic of China
  4. 4.Key Laboratory of Marine Bio-resources Sustainable Utilization (LMB-CAS), Guangdong Key Laboratory of Marine Materia Medica (LMMM-GD), South China Sea Institute of OceanologyChinese Academy of SciencesGuangzhouPeople’s Republic of China

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