True-Isothermal Plasmid Amplication System

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.