Isothermal Amplification for Detection of Nucleic Acids

In this proposal we discuss the development of a technique to amplify the signal generated when a labeled chimeric oligonucleotide hybridizes to it target sequence. Clearly there is a huge need for such a technique given the amount of variability that occurs during triditional amplification reactions, such as PCR. The chimeric oligonucleotide described in this proposal binds to it target sequence and becomes the subtrate for a specific endonuclease. Following degradation of the bound chimeric oligonucleotide, another oligonucleotide binds to the same sequence and this again becomes a target for degradation. Thus, the signal is amplified by many rounds of binding and degradation. The amplification and detection are performed entirely in solution in a single reaction tube with no reagent additions during the reaction. In Phase II of this study, we hope to develop this technology into a Kit that would be suitable to clinical applications, such as detecting bacterial or viral DNA in blood, plasma and other biological samples. PROPOSED COMMERCIAL APPLICATION: The successful completion of this study would result in a technology for amplifying a signal generated when a chimeric molecular beacon hybridizes to a target DNA in a mixed nucleic acid population. The commercial applications for this type of technology are vast including numerous clinical applications (diagnostic and prognostic) that would enable detection of particular viral (e.g. CMV or EBV), OR bacterial (e.g. Helicobacter Pylori, or Mycobacterium tuberculosis) sequences in human tissues or cell culture. Active Motif would make these reagents available to the research community.