Sun, 27 Jul 2008 10:14:30 BST CiteULike: vrich shotgun_cloning http://www.citeulike.org/user/vrich/tag/shotgun_cloning CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/vrich/article/2678872 <i>Appl. Environ. Microbiol., Vol. 72, No. 5. (1 May 2006), pp. 3291-3301.</i><br /><br />Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. 10.1128/AEM.72.5.3291-3301.2006 Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments Carl Abulencia Denise Wyborski Joseph Garcia Mircea Podar Wenqiong Chen Sherman Chang Hwai Chang David Watson Eoin Brodie Terry Hazen Martin Keller doi:10.1128/AEM.72.5.3291-3301.2006 Appl. Environ. Microbiol., Vol. 72, No. 5. (1 May 2006), pp. 3291-3301. 2008-04-16T20:11:54-00:00 2006 Appl. Environ. Microbiol. 72 5 3291 3301 mda phi29 shotgun_cloning