Linked Life Data

14657399

Source:http://linkedlifedata.com/resource/pubmed/id/14657399

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
26
pubmed:dateCreated
2003-12-24
pubmed:abstractText
We have improved upon the methodology and dramatically shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of bacteriophage X174 (5386 bp) from a single pool of chemically synthesized oligonucleotides. The procedure involves three key steps: (i). gel purification of pooled oligonucleotides to reduce contamination with molecules of incorrect chain length, (ii). ligation of the oligonucleotides under stringent annealing conditions (55 degrees C) to select against annealing of molecules with incorrect sequences, and (iii). assembly of ligation products into full-length genomes by polymerase cycling assembly, a nonexponential reaction in which each terminal oligonucleotide can be extended only once to produce a full-length molecule. We observed a discrete band of full-length assemblies upon gel analysis of the polymerase cycling assembly product, without any PCR amplification. PCR amplification was then used to obtain larger amounts of pure full-length genomes for circularization and infectivity measurements. The synthetic DNA had a lower infectivity than natural DNA, indicating approximately one lethal error per 500 bp. However, fully infectious X174 virions were recovered after electroporation into Escherichia coli. Sequence analysis of several infectious isolates verified the accuracy of these synthetic genomes. One such isolate had exactly the intended sequence. We propose to assemble larger genomes by joining separately assembled 5- to 6-kb segments; approximately 60 such segments would be required for a minimal cellular genome.
pubmed:commentsCorrections
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0027-8424
pubmed:author
pubmed:issnType
Print
pubmed:day
23
pubmed:volume
100
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
15440-5
pubmed:dateRevised
2009-11-18
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides.
pubmed:affiliation
Institute for Biological Energy Alternatives, 1901 Research Boulevard, Suite 600, Rockville, MD 20850, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't