Isothermal Accelerated Cascade Amplification

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 (lt0.5), 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.