权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

CASCADE ROLLING CIRCLE AMPLIFICATION FOR DNA DIAGNOSTICS

用于 DNA 诊断的级联滚环放大

基本信息

批准号：
2538806
负责人：
David D Thomas
金额：
$ 9.95万
依托单位：
ONCOR, INC.
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-09-30 至 1998-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2538806
关键词：
DNA chemical transfer reaction diagnosis design /evaluation diagnosis quality /standard fluorescence genetic markers macromolecule method development nucleic acid probes nucleic acid sequence rapid diagnosis thermodynamics

项目摘要

Many current nucleic acid amplification systems for detecting pathogenic agents may not yield consistent results because optimal amplification conditions vary with each target sequence. Cascade rolling circle amplification (CRCA) is a novel isothermal amplification system that uses generic primers to amplify circularized probes instead of the target. It is rapid, highly sensitive, and easily multiplexed, consistently yielding over a billion-fold amplification within one hour without using a thermocycler. If one of the primers is an energy transfer-labeled primer, homogeneous fluorescence detection of the CRCA product is possible within a closed system. The speed and ease of the assay make this technology suitable for field work or use in developing countries, as well as for simpler automation in diagnostic applications. The feasibility of this system has been demonstrated in preliminary experiments. Phase I objectives include optimization of ligation and amplification conditions to achieve maximum signal strength and sensitivity, and optimization of energy transfer-labeled primer structures that yield the highest fluorescence signal to background ratio without compromising amplification efficiency. The speed and sensitivity of the proposed method will be compared with traditional techniques using a pathogenic target Phase II studies will focus on clinical applications of this system. Proposed commercial applications: This amplification and detection system can form the basis of any DNA diagnostic procedure where the presence or absence of a specific nucleic and target sequence is determined, such as an infectious disease agent, inherited germline mutation, or somatic mutation in a cancer screening test The assay is highly sensitive, fast and simple, and we estimate that it will be less costly than current methods. Because it can be performed in a closed system, false-positive results from carry-over contamination will be minimized. The system is readily amenable to simple automation.

目前许多用于检测病原体的核酸扩增系统试剂可能不会产生一致的结果，因为最佳扩增条件随每个靶序列而变化。梯级滚圈 CRCA是一种新型的等温扩增系统，通用引物来扩增环化探针而不是靶。它快速、高灵敏度、易于多重检测，在一小时内扩增超过十亿倍，而不使用热循环仪如果引物之一是能量转移标记的引物， CRCA产品的均匀荧光检测是可能的，一个封闭的系统分析的速度和简便性使得这项技术适用于发展中国家的实地工作或使用，诊断应用中的自动化更简单。的可行性系统已在初步实验中得到证实。一期目的包括优化连接和扩增条件以实现最大的信号强度和灵敏度，并优化产生最高能量转移标记的引物结构荧光信号与背景比，而不损害扩增效率所提出的方法的速度和灵敏度将与使用致病靶点的传统技术相比研究将集中在该系统的临床应用上。拟议的商业应用：这种扩增和检测系统可以形成任何DNA的基础诊断程序，其中存在或不存在特定的核酸并确定靶序列，例如传染病病原体，遗传性生殖系突变或癌症筛查中的体细胞突变该检测方法灵敏度高，快速简便，我们估计，它将比目前的方法成本更低。因为它可以被执行在一个封闭的系统中，假阳性结果来自残留污染将被最小化。该系统易于实现简单的自动化。