Novel Convective Flow PCR Thermocycler

Texas Engineering Experiment Station, College Station TX

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Abstract

DESCRIPTION (provided by applicant): In order to address the ongoing need for rapid, inexpensive, portable, and reliable genomic analysis equipment, we propose a research effort to demonstrate that convective flow fields can be harnessed to perform the temperature cycling necessary for PCR amplification. A convective flow-based system operates by applying a temperature gradient across a flow network containing the PCR reagent mixture. This temperature gradient establishes a circulatory flow pattern that achieves thermal cycling by continuously transporting reagents through temperature zones associated with denaturation, annealing, and extension reactions. Since the convected reagents are in a continual state of thermal equilibrium with their surroundings, the thermal mass to be heated and cooled consists entirely of the fluid actively involved in the reaction. Consequently, convective flow PCR has the potential to achieve rapid cycling times in reactor volumes ranging from 50 nL to 50 mu L that integrate readily with existing laboratory protocols. By eliminating the need for dynamic external temperature control, a convective flow-based system is capable of achieving performance equal to or exceeding that of conventional thermocyclers in a greatly simplified format, This level of simplicity is a significant departure from previous attempts to construct novel thermocycling equipment, where added complexities often far outweigh any potential performance gains, We propose a research effort targeted at developing a new generation of thermocycling equipment offering improved performance at a significantly lower cost, thereby making PCR practical for use in a wider array of settings. This will be accomplished by achieving the following research Aims, Aim 1: Fully characterize the global 3-D velocity and temperature fields within convective flow reactors using coordinated particle image velocimetry, laser induced fluorescence, and computational simulations. Aim 2: Design, construct, and optimize a series of devices to perform convective flow-based PCR with the ability to achieve high throughput operation and integrate with existing laboratory protocols. Aim 3: Extend the use of convective-flow based systems to satisfy the needs of a variety of thermally activated biochemical reaction systems including RT-PCR, DNA cycle sequencing, and ligase detection.