Quantitative analysis of EDC-condensed DNA on vertically aligned carbon nanofiber gene delivery arrays
Article first published online: 8 DEC 2006
Copyright © 2006 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 97, Issue 4, pages 680–688, 1 July 2007
How to Cite
Mann, D. G.J., McKnight, T. E., Melechko, A. V., Simpson, M. L. and Sayler, G. S. (2007), Quantitative analysis of EDC-condensed DNA on vertically aligned carbon nanofiber gene delivery arrays. Biotechnol. Bioeng., 97: 680–688. doi: 10.1002/bit.21287
- Issue published online: 24 MAY 2007
- Article first published online: 8 DEC 2006
- Manuscript Accepted: 22 NOV 2006
- Manuscript Received: 29 AUG 2006
- National Institute for Biomedical Imaging and Bioengineering. Grant Number: 1-R01EB006316-01
- Laboratory Directed Research and Development funding program. Grant Number: 1-R21EB004066
- Material Sciences and Engineering Division Program. Grant Number: DE-AC05-00OR22725
- quantitative PCR;
Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. Polymerase chain reaction (PCR) and in vitro transcription (IVT) were used to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. This investigation shows the applicability of these methods for monitoring DNA immobilization techniques. Biotechnol. Bioeng. 2007;97: 680–688. © 2006 Wiley Periodicals, Inc.