Jump to main content
Jump to site search
Access to RSC content Close the message box

Continue to access RSC content when you are not at your institution. Follow our step-by-step guide.


Issue 12, 2010
Previous Article Next Article

A reliable method for detecting complexed DNA in vitro

Author affiliations

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.

Graphical abstract: A reliable method for detecting complexed DNA in vitro

Back to tab navigation

Supplementary files

Article information


Submitted
30 Jun 2010
Accepted
09 Aug 2010
First published
08 Sep 2010

Nanoscale, 2010,2, 2718-2723
Article type
Paper

A reliable method for detecting complexed DNA in vitro

C. Holladay, M. Keeney, B. Newland, A. Mathew, W. Wang and A. Pandit, Nanoscale, 2010, 2, 2718
DOI: 10.1039/C0NR00456A

Social activity

Search articles by author

Spotlight

Advertisements