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Article: Highly efficient deletion method for the engineering of plasmid DNA with single-stranded oligonucleotides

TitleHighly efficient deletion method for the engineering of plasmid DNA with single-stranded oligonucleotides
Authors
Issue Date2008
PublisherEaton Publishing Co. The Journal's web site is located at http://www.biotechniques.com
Citation
Biotechniques, 2008, v. 44 n. 2, p. 217-224 How to Cite?
AbstractThe λ phage Red recombination system has been used to modify plasmid, bacterial artificial chromosome (BAC), and chromosomal DNA in a highly precise and versatile manner. Linear double-stranded DNA fragments or synthetic single-stranded oligonucleotides (SSOs) with short flanking homologies (<50 bp) to the target loci can be used as substrates to direct changes, including point mutations, insertions, and deletions. In attempts to explore mechanistic bases under this recombination process, we and others have previously identified factors that influence SSO-mediated single base substitutions. In this report, we focus our study on SSO-mediated deletion on plasmids. We found that SSOs as short as 63 bp were sufficient to mediate deletion as long as 2 kb with efficiency higher than 1%. Strand bias was consistently observed, and SSOs with sequences identical to the nascent lagging strand during replication always resulted in higher efficiency. Unlike SSO-mediated single nucleotide substitution, homology on each side of SSO flanking the fragment to be deleted was important for successful deletion, and abolishing the host methyl-directed mismatch repair (MMR) system did not lead to detectable changes in deletion efficiency. Finally, we showed that by optimizing its design, SSO-mediated deletion was efficient enough to make it possible to manipulate plasmids without selectable markers.
Persistent Identifierhttp://hdl.handle.net/10722/90801
ISSN
2015 Impact Factor: 2.298
2015 SCImago Journal Rankings: 1.132
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLu, LYen_HK
dc.contributor.authorHuen, MSYen_HK
dc.contributor.authorTai, ACPen_HK
dc.contributor.authorLiu, DPen_HK
dc.contributor.authorCheah, KSEen_HK
dc.contributor.authorHuang, JDen_HK
dc.date.accessioned2010-09-17T10:08:35Z-
dc.date.available2010-09-17T10:08:35Z-
dc.date.issued2008en_HK
dc.identifier.citationBiotechniques, 2008, v. 44 n. 2, p. 217-224en_HK
dc.identifier.issn0736-6205en_HK
dc.identifier.urihttp://hdl.handle.net/10722/90801-
dc.description.abstractThe λ phage Red recombination system has been used to modify plasmid, bacterial artificial chromosome (BAC), and chromosomal DNA in a highly precise and versatile manner. Linear double-stranded DNA fragments or synthetic single-stranded oligonucleotides (SSOs) with short flanking homologies (<50 bp) to the target loci can be used as substrates to direct changes, including point mutations, insertions, and deletions. In attempts to explore mechanistic bases under this recombination process, we and others have previously identified factors that influence SSO-mediated single base substitutions. In this report, we focus our study on SSO-mediated deletion on plasmids. We found that SSOs as short as 63 bp were sufficient to mediate deletion as long as 2 kb with efficiency higher than 1%. Strand bias was consistently observed, and SSOs with sequences identical to the nascent lagging strand during replication always resulted in higher efficiency. Unlike SSO-mediated single nucleotide substitution, homology on each side of SSO flanking the fragment to be deleted was important for successful deletion, and abolishing the host methyl-directed mismatch repair (MMR) system did not lead to detectable changes in deletion efficiency. Finally, we showed that by optimizing its design, SSO-mediated deletion was efficient enough to make it possible to manipulate plasmids without selectable markers.en_HK
dc.languageengen_HK
dc.publisherEaton Publishing Co. The Journal's web site is located at http://www.biotechniques.comen_HK
dc.relation.ispartofBioTechniquesen_HK
dc.subject.meshBase Sequenceen_HK
dc.subject.meshDNA, Single-Stranded - geneticsen_HK
dc.subject.meshEscherichia colien_HK
dc.subject.meshEscherichia coli Proteins - metabolismen_HK
dc.subject.meshGenes, Reporteren_HK
dc.subject.meshGenetic Engineering - methodsen_HK
dc.subject.meshGenetic Markersen_HK
dc.subject.meshGenotypeen_HK
dc.subject.meshMolecular Sequence Dataen_HK
dc.subject.meshMutS DNA Mismatch-Binding Protein - metabolismen_HK
dc.subject.meshMutation - geneticsen_HK
dc.subject.meshOligonucleotides - geneticsen_HK
dc.subject.meshPlasmids - geneticsen_HK
dc.subject.meshSequence Deletionen_HK
dc.titleHighly efficient deletion method for the engineering of plasmid DNA with single-stranded oligonucleotidesen_HK
dc.typeArticleen_HK
dc.identifier.emailHuen, MSY:huen.michael@hku.hken_HK
dc.identifier.emailCheah, KSE:hrmbdkc@hku.hken_HK
dc.identifier.emailHuang, JD:jdhuang@hkucc.hku.hken_HK
dc.identifier.authorityHuen, MSY=rp01336en_HK
dc.identifier.authorityCheah, KSE=rp00342en_HK
dc.identifier.authorityHuang, JD=rp00451en_HK
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.2144/000112684en_HK
dc.identifier.pmid18330349-
dc.identifier.scopuseid_2-s2.0-40949110562en_HK
dc.identifier.hkuros144105-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-40949110562&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume44en_HK
dc.identifier.issue2en_HK
dc.identifier.spage217en_HK
dc.identifier.epage224en_HK
dc.identifier.isiWOS:000253620800017-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridLu, LY=8686996700en_HK
dc.identifier.scopusauthoridHuen, MSY=23004751500en_HK
dc.identifier.scopusauthoridTai, ACP=23981427700en_HK
dc.identifier.scopusauthoridLiu, DP=21934191400en_HK
dc.identifier.scopusauthoridCheah, KSE=35387746200en_HK
dc.identifier.scopusauthoridHuang, JD=8108660600en_HK

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