Article: Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli

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TitleEfficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli
AuthorsWong, QNY1
Ng, VCW1
Lin, MCM1
Kung, HF1
Chan, D1
Huang, JD1
Issue Date2005
PublisherOxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/
CitationNucleic Acids Research, 2005, v. 33 n. 6, p. 1-9 [How to Cite?]
DOI: http://dx.doi.org/10.1093/nar/gni059
Abstractλ-Red system-based recombinogenic engineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. Here, we report the use of a single selectable marker to enhance the usefulness of this approach. The strategy is to utilize the thymidylate synthase A (thyA) gene, which encodes an enzyme involved in the synthesis of thymidine 5′-triphosphate, for both positive and negative selection. With this approach, we successfully created point mutations in plasmid and bacterial artificial chromosome (BAC) DNA containing the mouse Col10a1 gene. The results showed that the thyA selection system is highly efficient and accurate, giving an average of >90% selection efficiency. This selection system produces DNA that is free from permanent integration of unwanted sequences, thus allowing unlimited rounds of modifications if required. © The Author 2005. Published by Oxford University Press. All rights reserved.
ISSN0305-1048
2011 Impact Factor: 8.026
2011 SCImago Journal Rankings: 1.542
DOIhttp://dx.doi.org/10.1093/nar/gni059
ISI Accession Number IDWOS:000228398400007
PubMed Central IDPMC1072810
ReferencesReferences in Scopus
DC Field
Value
dc.contributor.authorWong, QNY
dc.contributor.authorNg, VCW
dc.contributor.authorLin, MCM
dc.contributor.authorKung, HF
dc.contributor.authorChan, D
dc.contributor.authorHuang, JD
dc.date.accessioned2008-06-12T06:31:31Z
dc.date.available2008-06-12T06:31:31Z
dc.date.issued2005
dc.description.abstractλ-Red system-based recombinogenic engineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. Here, we report the use of a single selectable marker to enhance the usefulness of this approach. The strategy is to utilize the thymidylate synthase A (thyA) gene, which encodes an enzyme involved in the synthesis of thymidine 5′-triphosphate, for both positive and negative selection. With this approach, we successfully created point mutations in plasmid and bacterial artificial chromosome (BAC) DNA containing the mouse Col10a1 gene. The results showed that the thyA selection system is highly efficient and accurate, giving an average of >90% selection efficiency. This selection system produces DNA that is free from permanent integration of unwanted sequences, thus allowing unlimited rounds of modifications if required. © The Author 2005. Published by Oxford University Press. All rights reserved.
dc.description.naturepublished_or_final_version
dc.format.extent388 bytes
dc.format.mimetypetext/html
dc.identifier.citationNucleic Acids Research, 2005, v. 33 n. 6, p. 1-9 [How to Cite?]
DOI: http://dx.doi.org/10.1093/nar/gni059
dc.identifier.citeulike161569
dc.identifier.doihttp://dx.doi.org/10.1093/nar/gni059
dc.identifier.eissn1362-4962
dc.identifier.epage9
dc.identifier.hkuros98288
dc.identifier.isiWOS:000228398400007
dc.identifier.issn0305-1048
2011 Impact Factor: 8.026
2011 SCImago Journal Rankings: 1.542
dc.identifier.issue6
dc.identifier.openurl
dc.identifier.pmcidPMC1072810
dc.identifier.pmid15800210
dc.identifier.scopuseid_2-s2.0-28644449037
dc.identifier.spage1
dc.identifier.urihttp://hdl.handle.net/10722/48990
dc.identifier.volume33
dc.languageeng
dc.publisherOxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/
dc.publisher.placeUnited Kingdom
dc.relation.ispartofNucleic Acids Research
dc.relation.referencesReferences in Scopus
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License
dc.subject.meshDNA, Recombinant - genetics
dc.subject.meshEscherichia coli - enzymology - genetics - growth & development
dc.subject.meshGenetic Engineering - methods
dc.subject.meshRecombination, Genetic
dc.subject.meshThymidylate Synthase - genetics
dc.titleEfficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli
dc.typeArticle
Author Affiliations
  1. The University of Hong Kong