Quantitative analysis of EDC-condensed DNA on vertically aligned carbon nanofiber gene delivery arrays
- Univ. of Tennessee, Knoxville, TN (United States). Center for Environmental Biotechnology
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Engineering Science and Technology Division
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS)
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. We used polymerase chain reaction (PCR) and in vitro transcription (IVT) 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. In conclusion,this investigation shows the applicability of these methods for monitoring DNA immobilization techniques.
- Research Organization:
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Sciences (CNMS)
- Sponsoring Organization:
- National Inst. for Biomedical Imaging and Bioengineering; USDOE Office of Science (SC)
- DOE Contract Number:
- AC05-00OR22725; 1-R01EB006316-01; 1-R21EB004066
- OSTI ID:
- 1311210
- Journal Information:
- Biotechnology and Bioengineering, Vol. 97, Issue 4; ISSN 0006-3592
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
Similar Records
GENETIC MODIFICATION OF GIBBERELLIC ACID SIGNALING TO PROMOTE CARBON SEQUESTRATION IN TREE ROOTS AND STEMS
Final Report Construction of Whole Genome Microarrays, and Expression Analysis of Desulfovibrio vulgaris cells in Metal-Reducing Conditions