Novel Real Time Isothermal DNA Amplification/Detection

描述(申请人提供)：在这项提案中，我们将描述一种新的方法和仪器，用于实时、靶标特异的等温扩增和DNA定量，该方法不依赖于聚合酶链式反应，也不需要昂贵的荧光标记寡核苷酸引物。这种新方法使用发夹环链接体DNA扩增(HLCA)过程来扩增目标DNA序列，该过程在复制的最初几轮中将自启动的发夹寡核苷酸引物掺入到线性DNA中。随后的几轮复制是从这些发夹结构中启动的，以提供等温、不依赖于引物的串联DNA结构的扩增。如果完全实现，该方法将极大地节省成本，并在病原体和基因的快速定量方面具有广泛的适用性。第一阶段之前获得的数据显示，HLCA方法可以在60摄氏度的条件下使用BST聚合酶在一小时内扩增出微量的目标DNA。在第一阶段，我们将优化这项技术，用于我们计划的仪器，并更彻底地测试使用SYBR绿(或类似)双链DNA荧光标记染料对新合成的扩增DNA进行定量检测。由于这一过程将产生双链DNA分子，SYBR绿色染料可以直接加入到扩增反应中并附着到新形成的链上。该方法将允许我们在微滴定板读取器或其他单光束分光光度计中使用简单、廉价的可见光检测来实时定量扩增的DNA。该仪器和方法将显著降低病原体和基因的实时扩增/检测的成本和复杂性。根据我们在开发商业上成功的、廉价的和强大的DNA纯化仪器方面的丰富经验，我们相信每次检测的成本将低于0.50美元，而仪器的成本低于5500美元。这与目前的技术相比是有利的，每个样本的成本为5-15美元，每个仪器的成本为30,000-150,000美元。我们将利用现有的微滴定板读取器，对我们的检测方法进行改进，以处理多达96个样本。我们还将建造一个简化、坚固的现场单元，用于实时检测16个或更少的样本。我们的自动化DNA纯化方法的简化版本将被整合到实时扩增仪器中，使其能够像在病原体检测工作中遇到的那样从粗略样本开始。该产品具有7.5亿美元的市场潜力，适用于临床、研究和国防应用。

DESCRIPTION (provided by applicant): In this proposal, we will describe a new method and instrument for real-time, target-specific isothermal amplification and DNA quantitation that does not rely on the polymerase chain reaction and does not require expensive fluorescent labeled oligo primers. This new method amplifies the target DNA sequence using the "hairpin loop concatamer DNA amplification" (HLCA) process, which incorporates self-priming hairpin oligo DNA primers into linear DNA during the initial rounds of replication. Subsequent rounds of replication are primed from these hairpin structures to give isothermal, primer-independent amplification of concatameric DNA structures. The method, if fully realized, has tremendous cost saving and generalized applicability for rapid pathogen and gene quantitation. Data obtained prior to Phase I showed that the HLCA method could amplify trace amounts of target DNA using Bst polymerase at 60 degrees C in one hour. In Phase I, we will optimize this technology for use in our planned instrument and more thoroughly test the use SYBR green (or similar) double-strand DNA fluorescent label dyes for quantitative detection of the newly synthesized amplified DNA. Since the process will produce double-strand DNA molecules, SYBR green dye can be added directly to the amplification reaction and adhere to the newly formed chains. The method will allow us to use a simple, inexpensive visible light detection in a microtiter plate reader or other single beam spectrophotometer for real-time quantitation of the amplified DNA. The instrument and method will significantly reduce the cost and complexity of real-time amplification/detection of pathogens and genes. Based upon our extensive experience in the development of commercially successful, inexpensive and robust instruments for purification of DNA, we believe that the cost per assay will be less than $0.50 with an instrument costing less than $5500. This compares favorably to current technology costing $5-15 per sample and $30,000 to $150,000 per instrument. We will utilize an existing microtiter plate reader, modified for our detection method, for processing up to 96 samples. We will also construct a simplified, rugged field unit for real-time detection of 16 or less samples. An abbreviated version of our automated DNA purification method will be incorporated into the real-time amplification instrument, allowing it to begin with crude samples as one would encounter in pathogen detection work. The product has a $750 million market potential addressing clinical, research and defense applications.