Authors: T. Ray, A. Hatch, K. Lintecum, S-H Chao, C. Youngbull
Affilation: Arizona State University, United States
Pages: 85 - 88
Keywords: real-time PCR, high throughput, emulsion, continuous flow
We present the design and development of a continuous flow quantitative Polymerase Chain Reaction system, suitable for multi-analyte, high-throughput, semi-autonomous assays. This system operates in a continuous fashion and is able to detect many different primer probe combinations as is required for environmental analyses. Our design focus on a high-throughput system with re-usable components for low levels of consumables, automated fluid handling and low power consumption, that provides repeatable results with control over multiple assay parameters including sample volume, temperature, cycle time, and fluorophore. We use a two-phase, continuous flow, emulsion based system to reduce sample cross-talk and increase sample throughput. The sample injection port has the flexibility to form 30nL to 15µL sized reaction volumes, each functioning as an isolated micro-reactor able to detect a different gene sequence. This technique allows us to introduce multiple primers from a primer-bank to be mixed with different aliquots of the DNA sample that is then mixed hydro-dynamically and introduced into a continuous oil stream. The reagents then pass through teflon tubing wrapped 40 times around a continuous flow two temperature zone cylindrical thermocycler. The fluorescence from each channel is then detected using a fiber optic bundle connected to a 64 channel PMT.