Abstract
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.