title: Isolation of high molecular weight DNA from marine sponge bacteria for BAC library construction.
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.
authors.affiliation: LMB-CAS, LMMM-GD, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People's Republic of China.
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Ouyang
authors.forename: Shikun
authors.initials: S
authors.lastname: Dai
authors.forename: Lianwu
authors.initials: L
authors.lastname: Xie
authors.forename: M S
authors.initials: MS
authors.lastname: Ravi Kumar
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.forename: Huimin
authors.initials: H
authors.lastname: Sun
authors.forename: Danling
authors.initials: D
authors.lastname: Tang
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
mesh_terms: D000818:Animals; D001419:Bacteria / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015723:Gene Library; D010802:Phylogeny; D011161:Porifera / Q000382:microbiology*; D013559:Symbiosis
chemical_list: D004247:DNA
references.citation: Science. 2004 Apr 2;304(5667):66-74
references.pmid: 15001713
references.citation: Curr Opin Biotechnol. 2004 Jun;15(3):187-90
references.pmid: 15193325
references.citation: Antimicrob Agents Chemother. 2003 Aug;47(8):2518-25
references.pmid: 12878514
references.citation: Org Lett. 2000 Aug 10;2(16):2401-4
references.pmid: 10956506
references.citation: Methods. 2007 Aug;42(4):358-76
references.pmid: 17560324
references.citation: Antimicrob Agents Chemother. 1997 Jul;41(7):1521-30
references.pmid: 9210678
references.citation: Environ Microbiol. 2004 Sep;6(9):921-7
references.pmid: 15305917
references.citation: FEMS Microbiol Lett. 2003 Jun 6;223(1):15-20
references.pmid: 12798994
references.citation: Appl Environ Microbiol. 2000 Jun;66(6):2541-7
references.pmid: 10831436
references.citation: Trends Biotechnol. 2005 Nov;23(11):539-43
references.pmid: 16154653
references.citation: Microbiol Rev. 1983 Dec;47(4):621-69
references.pmid: 6363901
references.citation: Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7839-43
references.pmid: 7644502
references.citation: Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12830-5
references.pmid: 14566056
references.citation: Antonie Van Leeuwenhoek. 2008 Mar;93(3):241-8
references.pmid: 17717723
references.citation: Appl Environ Microbiol. 2005 Aug;71(8):4840-9
references.pmid: 16085882
references.citation: J Nat Prod. 2004 Aug;67(8):1314-8
references.pmid: 15332848
references.citation: Org Lett. 2001 Jun 28;3(13):1981-4
references.pmid: 11418029
references.citation: Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16222-7
references.pmid: 15520376
references.citation: Appl Environ Microbiol. 2007 Apr;73(7):2144-55
references.pmid: 17293531
references.citation: J Ind Microbiol Biotechnol. 2006 Jul;33(7):539-44
references.pmid: 16598493
references.citation: Microbiol Mol Biol Rev. 2004 Dec;68(4):669-85
references.pmid: 15590779
references.citation: Mar Biotechnol (NY). 2005 May-Jun;7(3):142-62
references.pmid: 15776313
references.citation: Extremophiles. 2004 Dec;8(6):475-88
references.pmid: 15252724
references.citation: Appl Environ Microbiol. 2001 Jan;67(1):434-44
references.pmid: 11133476
references.citation: Microb Ecol. 2002 Jan;43(1):134-45
references.pmid: 11984635
references.citation: Genomics. 1998 Aug 15;52(1):1-8
references.pmid: 9740665
references.citation: Mol Biol Evol. 1987 Jul;4(4):406-25
references.pmid: 3447015
references.citation: Environ Microbiol. 2006 Aug;8(8):1460-70
references.pmid: 16872408
references.citation: Int J Oncol. 2006 Dec;29(6):1381-8
references.pmid: 17088975
references.citation: J Ind Microbiol Biotechnol. 2006 Feb;33(2):121-8
references.pmid: 16261359
references.citation: Mol Biol Evol. 2007 Aug;24(8):1596-9
references.pmid: 17488738
references.citation: Appl Environ Microbiol. 1997 May;63(5):1721-4
references.pmid: 9143108
references.citation: J Ind Microbiol Biotechnol. 2004 Aug;31(7):335-44
references.pmid: 15257441
references.citation: Science. 1998 Feb 6;279(5352):879-82
references.pmid: 9452391
references.citation: J Nat Prod. 2003 Nov;66(11):1490-4
references.pmid: 14640525
references.citation: Appl Environ Microbiol. 2002 Sep;68(9):4301-6
references.pmid: 12200279
references.citation: Curr Microbiol. 2004 Jun;48(6):441-6
references.pmid: 15170241
references.citation: Appl Microbiol Biotechnol. 2007 May;75(1):11-20
references.pmid: 17318533
references.citation: J Microbiol Methods. 2005 Jul;62(1):1-11
references.pmid: 15823390
references.citation: Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9509-14
references.pmid: 10449723
references.citation: Experientia. 1988 Dec 1;44(11-12):1021
references.pmid: 3197807