True-Isothermal Plasmid Amplication System

描述（由申请人提供）：扩增和检测来自DMA和RNA的特定靶序列是诊断和生物医学研究的基本工具。我们设计了一种新的等温DNA扩增方法，该方法可用于扩增环状DNA分子，通过模仿DNA复制的性质。我们的方法，环状解旋酶依赖性扩增（c-HDA），使用DNA解旋酶分离双链DNA的两条链，然后使用DNA聚合酶复制靶序列。我们的c-HDA技术有几个优点。首先，这是一个真正的等温反应，整个扩增反应可以在一个温度下进行，因为解旋酶能够酶促分离双链体DNA，即使没有预先变性。这为开发快速简单的DNA诊断方法提供了潜力，这些方法可用于在护理点或现场检测含有环状质粒的病原体，如炭疽芽孢杆菌。第二，我们的方法可以扩增两侧有两个引物的特异性靶序列和整个环状DNA。质粒DNA的纯化和分析是DNA克隆、测序和筛选的重要步骤。如果成功开发，我们的c-HDA方法可以用作一个管/一步过程，以取代耗时和劳动密集型的细胞培养，纯化和分析质粒DNA的步骤。虽然我们已经确定了使用c-HDA扩增质粒DNA的可行性，但目前的HDA系统还不够稳健，无法在市场上商业化。技术风险障碍是1）长扩增时间（3-6小时）; 2）低灵敏度; 3）假阳性（非特异性扩增）。虽然风险和不确定性很高，但我们根据我们的初步研究和其他已发表的观察结果提出了良好的证据，证明通过拟议的研究进一步提高我们的技术优势以克服技术风险障碍是可行的。

DESCRIPTION (provided by applicant): Amplification and detection of specific target sequences from DMA and RNA is a fundamental tool for diagnostics and biomedical research. We have devised a new isothermal DMA amplification method which can be used to amplify circular DMA molecules, by mimicking nature in terms of DNA replication. Our method, circular Helicase-Dependent Amplification (c-HDA), uses a DNA helicase to separate two strands of a duplex DNA followed by a DNA polymerase to copy the target sequence. Our c-HDA technology has several advantages. First, it is a true-isothermal reaction and the entire amplification reaction can be carried out in one temperature since helicases are able to separate duplex DNA enzymatically even without prior denaturation. This offers potential for the development of rapid and simple DNA diagnostic methods that could be used to detect pathogens containing circular plasmids such as Bacillus anthracis at point-of-care or in the field. Second, our method can amplify both the specific target sequence flanked by two primers and the entire circular DNA. Plasmid DNA purification and analysis is an essential step for DNA cloning, sequencing and screening. If successfully developed, our c-HDA method may be used as a one-tube/one-step process to substitute time-consuming and labor-intensive steps of cell-culturing, purifying and analyzing plasmid DNA. Although we have established the feasibility of using c-HDA to amplify plasmid DNA the current HDA systems are not robust enough to be commercialized in marketplace. The technical risk barriers are 1) long amplification time (3-6 hours); 2) low sensitivity; 3) false - positive (non-specific amplification). Although the risk and uncertainty are high, we present good evidences based on our preliminary research and other published observations that it is feasible to further our technical advantage to over come technical risk barriers through proposed research.