Development of a one-step isothermal DNA amplification system for diagnostics

描述(由申请人提供)：依赖于特定序列的扩增来识别感兴趣的基因的分子诊断测试经常使用聚合酶链式反应(PCR)。要进行聚合酶链式反应，需要灵敏且通常昂贵的仪器进行热循环，这有助于通过耐热的DNA聚合酶进行热变性、引物退火和引物延伸。执行聚合酶链式反应的设备要求限制了它的便携性和在需要点诊断中的使用，例如在现场分析潜在的生物恐怖分子或在患者的床边使用。因此，需要一种易于使用的等温DNA扩增系统，该系统可以容易地用于在现场进行诊断测试。在拟议的研究中，我们描述了一个基于一个新的HDA平台的一步解旋酶依赖的扩增系统：极端嗜热HDA或eHDA系统。新的eHDA平台将允许在所有反应成分存在的情况下进行分析，而不会出现任何中断或可能的交叉污染，从而添加额外的试剂。一步法HDA分析将有助于开发用于临床诊断实验室的等温分析专用试剂和其他类型的诊断测试。第一阶段的研究将审查准备eHDA系统用于诊断测试的可行性，这将通过两个具体目标来解决。第一个具体目标将解决引物设计方面的问题，以提高eHDA性能和检测灵敏度。第二个具体目标将通过补充辅助蛋白来解决增加eHDA扩增产量的问题。辅助蛋白，如SSB蛋白和MutL，将被提纯并在eHDA中进行测试，以确定扩增特异性和敏感性的改善。最终的具体目标将审查与eHDA分析一起使用的最佳检测方法。一个合适的检测系统有可能提高检测的灵敏度，而不是标准的凝胶电泳观察到的灵敏度。第一阶段研究的结果将促进基于eHDA平台的一步等温诊断测试的开发。

DESCRIPTION (provided by applicant): Molecular diagnostic tests that rely on the amplification of specific sequences to identify genes of interest frequently use the polymerase chain reaction (PCR). To perform PCR, sensitive and, often, expensive instrumentation is required for thermocycling, which facilitates heat denaturation, primer annealing and primer extension by a thermostable DNA polymerase. The equipment requirement for performing PCR limits its portability and use in point-of-need diagnostics, for example in the field to analyze potential bioterrorism agents or at a patient's bedside. Thus, there is a need for an easy to use, isothermal DNA amplification system that can easily be used to perform diagnostic tests in the field. In the proposed research, we describe a one-step Helicase-Dependent amplification system based on a new HDA platform: extreme- thermophilic HDA or eHDA system. The new eHDA platform will allow assays to be performed in the presence of all reaction components without any interruptions or possible cross contamination to add additional reagents. A one-step HDA assay will facilitate the development of isothermal analytic specific reagents and other types of diagnostic tests to be used in clinical diagnostic laboratories. The phase I research will examine the feasibility of preparing the eHDA system for use in diagnostic tests, which will be addressed by two specific aims. The first specific aim will address primer design concerns to improve eHDA performance and detection sensitivity. The second specific aim will address increasing eHDA amplification yields by supplementing reactions with accessory proteins. Accessory proteins, such as SSB proteins and MutL, will be purified and tested in eHDA to determine improvements on amplification specificity and sensitivity. The final specific aim will examine optimal detection methods for use with eHDA assays. An appropriate detection system has the potential to improve the sensitivity of the assay beyond that which is observed by standard gel electrophoresis. Results from Phase I research will facilitate development of one- step, isothermal diagnostic tests based on the eHDA platform.