Novel Convective Flow PCR Thermocycler

新型对流 PCR 热循环仪

• 批准号: 6937213
• 负责人: Victor M Ugaz
• 金额: $ 17.5万
• 依托单位: TEXAS ENGINEERING EXPERIMENT STATION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-18 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6937213
• 关键词: bioengineering /biomedical engineering; biomedical equipment development; computer simulation; fluid flow; fluorescence; high throughput technology; ligase; nonblood rheology; nucleic acid sequence; polymerase chain reaction; portable biomedical equipment; protein quantitation /detection; temperature

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.

描述(由申请人提供)： 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，以证明可以利用对流流场来执行聚合酶链式反应扩增所需的温度循环。基于对流流动的系统通过在包含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，通过连续输送试剂通过与变性、退火和延伸反应相关的温度区域来实现热循环。由于对流试剂与其周围环境处于持续的热平衡状态，因此要加热和冷却的热质完全由参与反应的流体组成。因此，对流聚合酶链式反应有可能在反应堆体积从50毫升到50亩的范围内实现快速循环时间，L很容易与现有的实验室方案相结合。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极大简化的形式实现等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，在以前的尝试中，增加的复杂性往往远远超过任何潜在的性能收益，我们提议进行一项研究，旨在开发以显著较低的成本提供更高性能的新一代热循环设备，从而使聚合酶链式反应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， 目的1：利用坐标粒子图像测速仪、激光诱导荧光和计算模拟，全面表征对流反应堆内的全球三维速度场和温度场。 目的2：设计、建造和优化一系列基于对流的聚合酶链式反应装置，以实现高通量操作并与现有的实验方案集成。 目的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要，包括RT-PCR、DNA循环测序和连接酶检测。