Isothermal Accelerated Cascade Amplification

描述（由申请人提供）：我们最近发现了一种等温DNA扩增方案，与PCR相比，该方案有望在速度上取得突破性的改进。从理论上讲，它可以用C= C 0来描述。（K.t）N，其中C是扩增的靶DNA片段的浓度; C 0是初始靶DNA负载（拷贝/反应）; K是所用酶的转换因子（反应/min）; t是反应时间（min）; N是由扩增系统设计定义的因子，并且可以是任何正整数e1。新的扩增方案由在方程中定义为因子N的多个连续扩增反应组成，其中第一反应为第二反应提供模板，而第二反应反过来，整个扩增过程是“定向的”，因为例如在第二反应中产生的产物不能用作第一反应中的模板，但在第三反应中触发扩增。初步研究已经表明，增加N的数量可以显著加快扩增速度。K是一个非常有影响力的因素，因为它反映了扩增链中各个反应的速度（每分钟的产物）。初步研究显示，由于扩增中使用的关键酶的不寻常性质，K因子目前低得不可接受（<0.5）。提出了一种简单直接的方法来增加系数K值，这将是I期研究的重点。对其他潜在酶的分析表明，达到高达30-40的K值是可以实现的。这样的K值将实现我们的放大速度目标。一旦开发和优化，这种新的DNA扩增技术是唯一适合于异常快速的真实的时间检测的核酸。这一发现将应用于疾病连锁研究、群体分析（如单核苷酸多态性鉴定和基因分型）、表达谱分析和定量病原体鉴定的研究和临床环境。需要简单仪器和快速读数的现场应用也是有吸引力的应用。 公共卫生相关性：该项目的主要目标是开发一种新的DNA扩增方法，该方法既非常快速又等温，用于核酸序列检测和鉴定。I期研究的结果表明，这种新发现的等温扩增反应将成为人类基因组学和临床诊断的上级工具。我们正在开发的技术将在研究和临床环境中用于疾病联系研究，人口分析，基因分型，表达谱和病原体鉴定等应用。

DESCRIPTION (provided by applicant): We have recently discovered an isothermal DNA amplification protocol which promises breakthrough improvement in speed compared to PCR. Theoretically, it is described by C=C0.(K.t)N, where C is the concentration of the amplified target DNA fragment; C0 is the initial target DNA load (copy/reaction); K is a turnover factor (reaction/min) of the enzymes used; t is reaction time (min); and N is a factor defined by the amplification system design and can be any positive integer number e1. Estimates indicate that the new amplification reaction can be much faster than PCR, providing a targeted amplification power of ~1010 in 1-5 minutes or even less. The new amplification scheme is comprised of a number of consecutive amplification reactions defined in the equation as factor N wherein the first reaction supplies template for second reaction while the second reaction, in turn, provides template for the third reaction and so on. The overall amplification process is "direction oriented" in that the product produced, for example, in the second reaction cannot serve as a template in the first reaction but triggers amplification in the third reaction. Preliminary studies have already shown that increasing the number N significantly speeds up the amplification. K is a very influential factor since it reflects the speed of individual reactions (product per min) in the amplification chain. Preliminary studies revealed that factor K is currently unacceptably low (<0.5), due to an unusual property of a key enzyme used in the amplification. A simple and straightforward approach to increase the factor K values has been proposed and will be the focus of the Phase I study. Analysis of other prospective enzymes indicates that reaching K values up to 30-40 is achievable. Such K values would achieve our goals for amplification speed. Once developed and optimized, this new DNA amplification technology is uniquely suited for exceptionally fast real time detection of nucleic acids. The discovery will have application in both research and clinical settings for disease linkage studies, population analysis such as single nucleotide polymorphism identification and genotyping, expression profiling and quantitative pathogen identification. Field applications requiring simple instrumentation and rapid readouts are also attractive applications. PUBLIC HEALTH RELEVANCE: The main objective of this project is to develop a new DNA amplification method that is both exceptionally fast and isothermal, for the purpose of nucleic acid sequence detection and identification. Results of the Phase I study indicate that this newly discovered isothermal amplification reaction will become a superior tool in human genomics and clinical diagnostics. The technology that we are developing will be useful in both research and clinical settings for applications such as disease linkage studies, population analysis, genotyping, expression profiling and pathogen identification.