True-Isothermal Plasmid Amplication System

真正等温质粒扩增系统

基本信息

批准号：
6882829
负责人：
Huimin Kong
金额：
$ 9.82万
依托单位：
BIOHELIX CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-03-14 至 2006-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6882829
关键词：
bacteria biotechnology circular DNA diagnosis design /evaluation diagnosis quality /standard helicase microorganism classification nucleic acid amplification techniques nucleic acid purification nucleic acid sequence rapid diagnosis

项目摘要

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.

描述（由申请人提供）：从 DMA 和 RNA 中扩增和检测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 DMA 扩增方法，通过模仿 DNA 复制的性质，可用于扩增环状 DMA 分子。我们的方法是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 聚合酶复制目标序列。我们的 c-HDA 技术具有多项优势。首先，它是真正的等温反应，并且整个扩增反应可以在一个温度下进行，因为即使没有事先变性，解旋酶也能够通过酶促方式分离双链 DNA。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，例如炭疽杆菌。其次，我们的方法可以扩增两侧引物侧翼的特定靶序列和整个环状 DNA。质粒DNA纯化和分析是DNA克隆、测序和筛选的重要步骤。如果开发成功，我们的 c-HDA 方法可作为一管/一步流程来替代细胞培养、纯化和分析质粒 DNA 等耗时且劳动密集的步骤。尽管我们已经确定使用 c-HDA 扩增质粒 DNA 的可行性，但当前的 HDA 系统还不够强大，无法在市场上商业化。技术风险壁垒是1）放大时间长（3-6小时）； 2）灵敏度低； 3）假阳性（非特异性扩增）。尽管风险和不确定性很高，但根据我们的初步研究和其他已发表的观察结果，我们提供了充分的证据，表明通过拟议的研究进一步增强我们的技术优势以克服技术风险障碍是可行的。