SBIR Phase II: Reagent Development for a Rapid Enzymatic DNA Synthesis Platform

点击翻译按钮获取中文摘要

The broader impact of this Small Business Innovation Research (SBIR) Phase II project is to optimize a system to rapidly synthesize long, user-defined sequences of DNA as a commercial service. The process will be capable of producing error-free DNA fragments, each long enough to encode multiple genes, in 1-2 weeks, at a price that will be affordable even to university research labs. Researchers currently can purchase short DNA fragments that must be stitched together to make functional genes – which is labor-intensive and sometimes impossible – or pay vendors to do it. The ability to buy an intact group of genes on one piece of DNA will help deepen the understanding of all biological systems, from animals and plants to the bacteria and viruses that infect them. Easy access to long DNAs will also allow synthetic biologists to build completely novel biological devices, such as bacteria that manufacture vitamins or medicines, cells that detect and destroy cancer, or new sustainable food ingredients and novel biomaterials.The proposed project is focused on developing an enzymatic method for DNA synthesis that will alleviate several problems inherent to chemical DNA synthesis, the only method currently available commercially. Chemical synthesis works well for short DNA fragments, but high-quality synthesis is limited to 200 bases. Longer fragments often are created by stitching together many short fragments, but this process is unreliable for sequences that contain repeats or high AT or GC content. In the proposed enzymatic DNA synthesis method, a single deoxynucleoside triphosphate (dNTP) is conjugated to the template-independent polymerase Terminal Deoxynucleotidyl Transferase (TdT) by a cleavable linker. When presented with a DNA primer, the dNTP-TdT conjugate adds its tethered nucleotide and remains attached, blocking further elongation by other conjugates. Cleavage of the linker releases the TdT, to be washed away with unreacted conjugates, and exposes the oligo for extension with the next dNTP-TdT conjugate. These two steps of “extension” and “deprotection” are iterated to synthesize a defined sequence. The accuracy of the resulting DNA strand depends on the stability and cleavage efficiency of the linker. A variety of linkages and cleavage enzymes will be tested to yield 99.9% stability and cleavage in rapid, 30-second reactions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.