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）：DMA和RNA的特定目标序列的扩增和检测是诊断和生物医学研究的基本工具。我们已经设计了一种新的等温DMA扩增方法，该方法可用于通过模仿DNA复制的性质来扩增圆形DMA分子。我们的方法是圆形解旋酶依赖性扩增（C-HDA），使用DNA解旋酶分离双链DNA的两条链，然后是DNA聚合酶，以复制靶序列。我们的C-HDA技术具有几个优势。首先，它是一个真实的反应，并且可以在一个温度下进行整个放大反应，因为解放酶即使没有事先变性也能够在酶促上分离酶促的双链DNA。这为开发快速而简单的DNA诊断方法提供了潜力，该方法可用于检测含有圆形质粒的病原体，例如在护理点或现场中的炭疽芽孢杆菌。其次，我们的方法可以扩增两个引物和整个圆形DNA的特定靶序列。质粒DNA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方法可以用作单管/一步过程，以替代细胞培养，纯化和分析质粒DNA的耗时和劳动密集型步骤。尽管我们已经确定了使用C-HDA扩增质粒DNA的可行性，但当前的HDA系统不足以在市场上商业化。技术风险障碍是1）长放大时间（3-6小时）； 2）低灵敏度； 3）假阳性（非特异性扩增）。尽管风险和不确定性很高，但我们根据初步研究和其他发表的观察结果提出了良好的证据，即通过拟议的研究来进一步越来越多地通过技术风险障碍来实现我们的技术优势，这是可行的。