Linked Life Data

16286637

Source:http://linkedlifedata.com/resource/pubmed/id/16286637

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
48
pubmed:dateCreated
2005-11-30
pubmed:abstractText
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.
pubmed:commentsCorrections
http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-10572186, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-10731132, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-11381035, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-11808696, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-12384570, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-14657399, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-14988392, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-16023891, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-16030319, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-16056220, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-16163333, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-1733957, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-2498321, http://linkedlifedata.com/resource/pubmed/commentcorrection/16286637-8428945
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0027-8424
pubmed:author
pubmed:issnType
Print
pubmed:day
29
pubmed:volume
102
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
17332-6
pubmed:dateRevised
2009-11-18
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Cell-free cloning using phi29 DNA polymerase.
pubmed:affiliation
Synthetic Biology Group, The J. Craig Venter Institute, Rockville, MD 20850, USA. chutchison@venterinstitute.org
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't