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dc.contributor.authorKuhn, Heikoen_US
dc.contributor.authorFrank-Kamenetskii, Maxim D.en_US
dc.date.accessioned2012-01-11T00:39:16Z
dc.date.available2012-01-11T00:39:16Z
dc.date.copyright2008
dc.date.issued2008-4
dc.identifier.citationKuhn, Heiko, Maxim D. Frank-Kamenetskii. "Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases" Nucleic Acids Research 36(7): e40. (2008)
dc.identifier.issn1362-4962
dc.identifier.urihttps://hdl.handle.net/2144/3028
dc.description.abstractWe describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3′ terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3′-flap DNA is obtained. The single-stranded terminus in 3′-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic λ-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.en_US
dc.description.sponsorshipWallace A. Coulter Foundationen_US
dc.language.isoen
dc.publisherOxford University Pressen_US
dc.rightsCopyright 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.en_US
dc.rights.urihttp://creativecommons.org/licenses/by-nc/2.0/uk/
dc.titleLabeling of Unique Sequences in Double-Stranded DNA at Sites of Vicinal Nicks Generated by Nicking Endonucleasesen_US
dc.typeArticleen_US
dc.identifier.doi10.1093/nar/gkn107
dc.identifier.pmid18344522
dc.identifier.pmcid2373456


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Copyright 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Except where otherwise noted, this item's license is described as Copyright 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.