(211j) Continuous In Vitro Transcription of H5 Influenza Vrna In a Novel Microreactor

Biological molecules such as RNA or DNA are often present in contaminated clinical samples and in low concentrations, creating a need for both separation and amplification. Immobilization of RNA and DNA on solid substrates allows researchers to isolate and purify dilute molecules during transcription and amplification reactions while recycling rare DNA templates. When compared to the solution phase, however, immobilized DNA and RNA directed reactions are less efficient due to steric interference and mass transfer limitations; a fact that is particularly noticeable when working with large molecules. We propose to overcome these limitations by transferring an in vitro transcription reaction into a novel glass microreactor where solution conditions can be carefully controlled. A constriction in the reactor channel immobilizes glass beads functionalized with 1814bp H5 Influenza cDNA molecules, which serve as a template for RNA production using an SP6 RNA polymerase. By varying applied pressure, we can control the flow rate of reagents and enzyme while simultaneously removing harmful reaction products. An additional downstream channel mixes molecular beacons or Ribogreen for real time detection of RNA production. The potential applications of this system are broad, ranging from amplification of rare mRNA specimens for cellular expression assays to RNA amplification for a sequence specific disease detection assay. Ideally, this system can produce RNA more efficiently than both the solution phase and substrate-immobilized in vitro transcription reactions.