The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to improve the ability to amplify very long DNA in a few hours with high precision and few errors. The polymerase chain reaction amplification technology invented 30 years ago has revolutionized biomedical sciences. The proposed 100 kilobase (Kb) DNA amplification technology is ten times longer than existing methods, which would enable multiple technical and commercial capabilities not currently possible. These new tools will improve human health via high resolution sequence analysis of cancer genomes, safer DNA therapeutic applications, more rapid natural product drug discovery, and simplified genome engineering for stem cell biology research. Furthermore, this technology has the potential to revolutionize scientific and commercial fields such as oncology, transplantation, microbiology, gene therapy, synthetic biology, drug discovery, and basic molecular and cellular biology. The economic impact of the proposed research in the form of marketable products and services also is substantial. It is estimated that the patent protectable 100 kb DNA amplification technologies and applications could generate over $1B in revenues over the 20-year life span of the various products in four markets: Amplification, sequencing, gene therapy, and synthetic biology.This SBIR Phase I project proposes to develop 100 kb DNA amplification tools that dramatically overcome the commercial and technical limits of current in vitro DNA amplification products. Complex problems in biomedical sciences remain prohibitively costly, time-consuming, and laborious due to long-range sequence information that is lost or scrambled using existing short-range amplification tools. Research products that can amplify anonymous and sequence-specific DNA in long contiguous stretches approaching 100 Kb are needed for improvements in sequence analysis, cloning, mapping, transfection, expression, and related biotechnology applications. The research goal is to develop novel enzymatic approaches to amplify long stretches of DNA utilizing a native consortium of high fidelity replication enzymes, as opposed to single enzyme systems. The plan is to build on existing resources that have proven superior for the replication of long DNA, however, using new methods that dramatically reduce the side reactions and problems inherent with single enzyme systems. A scientific renaissance surrounding long-range amplification and sequencing methods is envisioned that radically simplifies the sequencing and assembly of cancer genome rearrangements, engineering large, complex metabolic pathways encoding drugs, and producing long tracts of vector- and virus-free DNA for gene therapy and stem cell induction.
