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  • A reliable method for detecting complexed DNA in vitro

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    Author(s)
    Holladay, C.
    Keeney, M.
    Newland, B.
    Mathew, Asha
    Wang, W.
    Pandit, A.
    Griffith University Author(s)
    Mathew, Asha
    Year published
    2010
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    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 ...
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    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ssay only allowed detection of three types of complexes (those formed using Lipofectin頡nd 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鮠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.
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    Journal Title
    Nanoscale
    Volume
    2
    Issue
    12
    DOI
    https://doi.org/10.1039/c0nr00456a
    Copyright Statement
    © 2010 Royal Society of Chemistry. This is the author-manuscript version of this paper. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal website for access to the definitive, published version.
    Subject
    Chemical Sciences not elsewhere classified
    Physical Sciences not elsewhere classified
    Technology not elsewhere classified
    Physical Sciences
    Chemical Sciences
    Technology
    Publication URI
    http://hdl.handle.net/10072/64705
    Collection
    • Journal articles

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