Novel Convective Flow PCR Thermocycler

描述（由申请人提供）： 为了解决快速，廉价，便携式和可靠的基因组分析设备的持续需求，我们提出了一项研究工作，以证明对流场可以利用PCR扩增所需的温度循环。基于对流的系统通过在包含PCR试剂混合物的流动网络上施加温度梯度来操作。该温度梯度建立了循环流动模式，其通过将试剂连续输送通过与变性、退火和延伸反应相关的温度区来实现热循环。由于对流反应物与其周围环境处于连续的热平衡状态，因此待加热和冷却的热物质完全由积极参与反应的流体组成。因此，对流PCR具有在反应器体积范围从50 nL到50 μ L中实现快速循环时间的潜力，其容易与现有的实验室方案整合。通过消除对动态外部温度控制的需要，基于对流流动的系统能够以大大简化的形式实现等于或超过常规热循环仪的性能。这种简单性水平显著偏离了构建新型热循环设备的先前尝试，其中增加的复杂性通常远远超过任何潜在的性能增益。我们提出了一项研究工作，旨在开发新一代的热循环设备，以显着降低成本，提供更好的性能，从而使PCR实际用于更广泛的设置。这将通过实现以下研究目标来实现， 目标1：使用协调粒子图像测速、激光诱导荧光和计算模拟全面表征对流反应器内的全局三维速度场和温度场。 目标二：设计，构建和优化一系列设备，以执行基于对流的PCR，能够实现高通量操作并与现有实验室方案集成。 目标3：扩展基于对流的系统的使用，以满足各种热激活生化反应系统的需求，包括RT-PCR、DNA循环测序和连接酶检测。

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.