Novel Real Time Isothermal DNA Amplification/Detection

新型实时等温 DNA 扩增/检测

• 批准号: 6641930
• 负责人: WILLIAM P MACCONNELL
• 金额: $ 10万
• 依托单位: MACCONNELL RESEARCH CORPORATION
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-03 至 2003-09-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6641930
• 关键词: DNA primers; biotechnology; method development; nucleic acid amplification techniques; nucleic acid quantitation /detection; nucleic acid structure; thermography; time resolved data

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.

描述（由申请人提供）：在本提案中，我们将描述一种用于实时、靶特异性等温扩增和DNA定量的新方法和仪器，该方法和仪器不依赖于聚合酶链反应，也不需要昂贵的荧光标记寡核苷酸引物。这种新方法使用“发夹环多联体DNA扩增”（HLCA）过程扩增靶DNA序列，该过程在最初几轮复制期间将自引发发夹寡聚DNA引物掺入线性DNA中。随后的几轮复制从这些发夹结构引发，以产生多联体DNA结构的等温、引物非依赖性扩增。该方法，如果完全实现，具有巨大的成本节约和快速病原体和基因定量的普遍适用性。在第一阶段之前获得的数据表明，HLCA方法可以在60摄氏度下使用Bst聚合酶在一小时内扩增痕量的靶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绿色（或类似）双链DNA荧光标记染料定量检测新合成的扩增DNA。由于该过程将产生双链DNA分子，SYBR绿色染料可以直接加入到扩增反应中并粘附到新形成的链上。该方法将使我们能够使用一个简单的，廉价的可见光检测在微量滴定板阅读器或其他单光束分光光度计的实时定量扩增的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。基于我们在开发商业上成功的、廉价的和稳健的DNA纯化仪器方面的丰富经验，我们相信每次测定的成本将低于0.50美元，仪器成本低于5500美元。这与目前每个样品5 -15美元和每个仪器30，000 - 150，000美元的技术相比是有利的。我们将利用现有的微量滴定板阅读器，为我们的检测方法进行修改，处理多达96个样本。我们还将建造一个简单、坚固的现场单元，用于实时检测16个或更少的样品。我们的自动化DNA纯化方法的缩写版本将被纳入实时扩增仪器中，使其能够像病原体检测工作中遇到的那样从粗样品开始开始。该产品具有7.5亿美元的市场潜力，可用于临床、研究和国防应用。