This Small Business Innovation Research (SBIR) Phase II project proposes to develop novel products for routine genetic testing by demonstrating feasibility of an innovative technology called Autoligation Chain Reaction (ACR). The intellectual merit of ACR is an enabling nucleic acid amplification technology that requires no nucleotides or enzymes. Polymerase inhibitors are found in many laboratory samples and clinical specimens, and contribute to the high cost of molecular-based assays in routine genetic tests because labor-intensive sample preparation and assay development are required to optimize around these inhibitors with current molecular technologies. Because ACR does not involve any reagents sensitive to polymerase inhibitors, the requirement for sample preparation is expected to be low and overall assay development and testing turnaround times are expected to be much faster. Specific key research objectives of the project include design and synthesis of thermal-stable ACR reagents, and the demonstration that ACR can exponentially amplify DNA target sequences without enzymes or nucleotides. Research will be carried out using low copy-number target nucleic acid sequences containing bio-relevant SNPs across multiple loci. The anticipated technical results should show robust, specific, and reproducible amplifications of multiple SNPs on multiple loci in the absence of enzymes or nucleotides.
What are the broader impacts of the proposed activity?
The broader impact/commercial potential of this project, if successful, is the innovation of an enabling technology that could dramatically reduce sample preparation and assay optimization times, and significantly increase the efficiency and quality, and lower the cost of clinical diagnostics and routine genetic testing. Since non-enzymatic amplification coupled with the inherent simplicity of ACR makes this technology more amenable to standardization in clinical and lab settings across different sample types than existing molecular technologies, it is expected that ACR technology will drive the development of a new generation of molecular diagnostic and screening products. These products are expected to provide more efficient, simpler, cheaper, faster, and accurate routine genetic testing of a broad range of biomarkers for a wide range of diseases and genetic disorders, including those currently unattainable by traditional molecular methods. As a result, ACR will potentially not only advance the understanding of diseases at the genetic level, but also bring broader benefits to human health and society at large through enhanced biomedical discovery, diagnostics, and personalized medicine.
1. Intellectual Merits Summary: This Small Business Innovation Research (SBIR) Phase II project developed novel assay products for SNP biomarker screening using an innovative nucleic acid amplification technology platform. The intellectual merits are that no enzymes, nucleotides, probes, master mixes, extension reactions, or sample preparations were used to amplify target nucleic acid sequences, in both single-plex and multiplex. Accomplishing the stated research objectives in this proposal resulted in a universal assay technology platform that is more cost-effective, easier-to-use, and more readily scalable with the needs of researchers involved in routine genetic testing. A summary of the intellectual merits includes: High assay reagent and sample optimization costs make polymerase chain reaction (PCR) technology too expensive for routine genetic testing, and PCR-based technologies are susceptible to enzyme inhibitors and have limits of detection (LOD) that are not amenable to the detection of rare mutations in some diseases in heterogeneous samples. The technology platform developed during this Phase II project required no upfront sample preparation, and no additional optimization of multiplexing reactions. Furthermore, assays manufactured based on this technology platform demonstrated robust amplification of nucleic acid target sequences without enzymes, nucleotides, probes, master mixes, or extension reactions, and were shown to be resistant to inhibitors found in unpurified extracts that inhibited PCR. Results from this Phase II project also suggest an LOD of 10-6, or lower, with significantly greater cost effectiveness to manufacture the novel assays, making this technology platform more amenable to routine early detection of disease biomarkers when therapeutic interventions are much more effective. 2. Broader Impacts: The broader impact/commercial potential of this project is an enabling next-generation amplification technology platform with a broader impact potential to i) enable new applications and shift paradigms for routine molecular testing, ii) enhance selectivity and portability for earlier disease detection and stratification when therapeutic interventions are much more effective, and iii) enable routine point-of-care clinical testing to improve clinical triage decisions, routine monitoring, and treatment outcomes. This new technology platform will potentially not only advance our understanding of diseases at the genetic level, but also bring broader benefits to human health and society at large through enhanced biomedical discovery, diagnostics, and personalized medicine in a more cost-effective and easier-to-use manner. Commercial Impacts: The technology platform is anticipated to enable selective, rapid, and affordable biomarker detection without having to invest in labor-intensive sample preparation procedures. The envisioned products will be assays, panels, and kits for the detection of plant traits and disease biomarkers in routine genetic testing settings, with the objectives of providing more cost-effective and easier-to-use alternatives for routine genetic testing.
