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A reliable method for detecting complexed DNA in vitro

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dc.contributor.author Holladay, C. en
dc.contributor.author Keeney, M. en
dc.contributor.author Mathew, B en
dc.contributor.author Wang, W. en
dc.contributor.author Pandit, A. en
dc.date.accessioned 2010-10-14T09:21:00Z en
dc.date.available 2010-10-14T09:21:00Z en
dc.date.issued 2010-09-08 en
dc.identifier.citation Holladay, C., Keeney, M., Newland, B., Mathew, A., Wang, W., & Pandit, A. A reliable method for detecting complexed DNA in vitro. Nanoscale. en
dc.identifier.issn 2040-3364 en
dc.identifier.uri http://hdl.handle.net/10379/1337 en
dc.description.abstract Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique hypothesized to allow quantification of both bound and unbound DNA. A comparison of the two methods was performed by quantifying the elution of six different varieties of DNA complexes from a model biomaterial (collagen) scaffold. After seven days of elution, the PicoGreen® assay only allowed detection of three types of complexes (those formed using Lipofectin¿ and two synthesised copolymers). However, the Cy5 fluorescent labeling technique enabled detection of all six varieties including those formed via common transfection agents poly(ethylene imine), poly-l-lysine and SuperFect¿. This allowed reliable quantification of the elution of all these complexes from the collagen scaffold. Thus, while intercalating dyes may be effective and reliable for detecting double-stranded, unbound DNA, the technique described in this work allowed reliable quantification of DNA independent of complexation state. en
dc.format application/pdf en
dc.publisher The Royal Society of Chemistry en
dc.subject Complexed nucleic acids en
dc.subject Release study en
dc.subject Gene delivery en
dc.subject Transfection en
dc.subject Network of Excellence for Functional Biomaterials en
dc.title A reliable method for detecting complexed DNA in vitro en
dc.type Article en
dc.local.publishedsource http://dx.doi.org/10.1039/C0NR00456A en
dc.description.peer-reviewed peer-reviewed en
dc.contributor.funder Science Foundation Ireland en

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