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. ? ? ?
| Goldmeyer, James; Kong, Huimin; Tang, Wen (2007) Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection. J Mol Diagn 9:639-44 |
