Sat, 19 Jul 2008 02:18:09 BST CiteULike: de vrich phi29 http://www.citeulike.org/user/vrich/tag/phi29 CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/vrich/article/702191 <i>Proc Natl Acad Sci U S A, Vol. 102, No. 48. (29 November 2005), pp. 17332-17336.</i><br /><br />We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5-7 kb in size by >10(9)-fold, using phi29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using phi29 DNA polymerase is "background" DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using phi29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coli, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the phi29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using phi29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopolymer tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning. Cell-free cloning using phi29 DNA polymerase. CA Hutchison HO Smith C Pfannkoch JC Venter doi:10.1073/pnas.0508809102 Proc Natl Acad Sci U S A, Vol. 102, No. 48. (29 November 2005), pp. 17332-17336. 2006-06-20T11:16:35-00:00 2005 Proc Natl Acad Sci U S A 0027-8424 102 48 17332 17336 mda phi29 http://www.citeulike.org/user/vrich/article/694536 <i>Nature Biotechnology, Vol. 24, No. 6. (28 May 2006), pp. 680-686.</i> Sequencing genomes from single cells by polymerase cloning Kun Zhang Adam Martiny Nikos Reppas Kerrie Barry Joel Malek Sallie Chisholm George Church doi:10.1038/nbt1214 Nature Biotechnology, Vol. 24, No. 6. (28 May 2006), pp. 680-686. 2006-06-13T09:43:51-00:00 2006 Nature Biotechnology 1087-0156 24 6 680 686 Nature Publishing Group phi29 prochlorococcus single_cell_genomics 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