Novel Convective Flow PCR Thermocycler

超出所提供的空间。为了满足对快速、廉价、便携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，以证明对流流场可以用来进行PCR扩增所需的温度循环。基于对流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。这种温度梯度建立了一种循环流动模式，通过与变性、退火和延伸反应相关的温度区连续输送试剂，实现热循环。由于对流试剂与其周围环境处于持续的热平衡状态，因此被加热和冷却的热质量完全由积极参与反应的流体组成。因此，对流流式PCR有可能在反应器体积范围从50 nL到50 FL内实现快速循环时间，并且很容易与现有的实验室方案集成。通过消除对动态外部温度控制的需求，基于对流流的系统能够以大大简化的格式实现等同于或超过传统热循环器的性能。这种简单性水平与以前构建新型热循环设备的尝试有很大不同，在这种设备中，增加的复杂性通常远远超过任何潜在的性能收益。我们提出了一项研究工作，旨在开发新一代热循环设备，以显着降低成本提高性能，从而使PCR在更广泛的环境中实际使用。这将通过实现以下研究目标来实现：目标1：利用协调粒子图像测速、激光诱导荧光和计算模拟来全面表征对流反应器内的全局三维速度和温度场。目标2：设计，构建和优化一系列设备，以执行基于对流流的PCR，具有实现高通量操作的能力，并与现有实验室协议集成。目标3：扩展对流系统的使用，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性能SITE ========================================Section End===========================================

EXCEED THE SPACE PROVIDED. 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 FL 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 PERFORMANCE SITE ========================================Section End===========================================