DNA and
RNA are commonly captured on solid substrates during purification and isolation, where they can be transferred to downstream amplification and transcription reactions. When compared to the
solution phase, however,
immobilized DNA- and
RNA-directed reactions are less efficient because of a variety of complex factors. Steric inhibition because of the bead surface and neighboring
biological polymers, a change in
solution chemistry because of the high local concentration of template molecules, and mass transfer to the bead surface could all affect the overall reaction kinetics. Furthermore, these effects may be particularly evident when working with long clinically relevant molecules, such as
mRNA,
viral RNA, and
cDNA. In this paper, we focus on the in vitro transcription reaction (IVT) of both a long and short strand of H5
influenza A
RNA (1777 and 465 nt) on both free and
immobilized DNA templates to study these phenomena. We found that transcription was less efficient on immobilized beads than in
solution, but that it can be dramatically increased with optimal
solution chemistry. Using high
ribonucleotide concentrations (>6 mM total rNTP), the
RNA yield from long immobilized
cDNA templates was boosted to 60% of
solution control. Surprisingly, we found that steric effects because of surrounding immobilized molecules were only significant when the
DNA molecules were short enough to achieve a high density (9x10(-4) microm2/molecule) on the
silica substrate, such that the gap between molecules is on the order of the polymerase diameter. Eventually, these findings can be exploited in an automated microreactor, where isolation, purification, amplification, and detection of
nucleic acids can be unified into one portable device.