Steric effects and mass-transfer limitations surrounding amplification reactions on immobilized long and clinically...

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.