The purpose of the present project is to develop and validate a new method for synthesizing large number of DNA fragments, oligodeoxynucleotides. The method, which is called "sequence-directed DNA synthesis", involves the use of small electronic circuits, "p-Chips", each of which transmits a unique ID number via RF when illuminated with laser light. In a massively parallel format, oligonucleotides are synthesized using p-Chips as the solid phase. The p-Chip ID provides the ability to associate a given DNA sequence with that particular p-Chip as synthesis proceeds. A key part of the system is a high-speed instrument, the sorter, that reads the ID of each p-Chip and then sorts p-Chips into the appropriate reaction vessel based on the p-Chip ID, thus the specific DNA sequence. The system, when completed, will be able to generate synthetic DNA fragments in very short times and at costs that are much lower than current methods. The work plan for the project is to initially, in Phase I, demonstrate the ability to synthesize DNA on currently available 0.5 mm p-Chips and to show feasibility of sorting. Following this, in Phase II, will shift to a new p-Chip design, each of which is one thousandth the volume of the 0.5 mm p-Chip. The main goals will be to build the sorter for these ultra-small chips, and to formulate a working method for large-scale sequence-directed combinatorial DNA synthesis. We will also demonstrate applicability of the method by synthesizing a large number of oligonucleotides on p-Chips and creating a 10 kb long gene fragment. We will obtain the DNA sequence error rate upon cloning and identify the best methods for gene assembly and construction of long segments of chromosomes. The prospect of rapidly producing DNA at low prices has profound implications in a number of fields related to synthetic genomics where new tools are needed. This includes the design of modified genes for enzymes, energy production, vaccine designs and many other applications.
Public Health Relevance Statement: Public Health Relevance: Synthetic genomics is projected to be a rapidly developing field in molecular genetics. PharmaSeq's sequence-directed DNA synthesis will offer a powerful tool for scientists to design and build many variants of genes and larger genomic fragments at costs that are far lower than those today. The ability to economically design new DNA-based products using PharmaSeq's sorting technology will accelerate research in diagnostics, drug discovery and applied chemistry and provide innovative products.
NIH Spending Category: Bioengineering; Biotechnology; Genetics; Human Genome
Project Terms: Award; base; Chemistry; Chromosomes; Cloning; combinatorial; Combinatorial Synthesis; Computers; cost; Custom; design; Diagnostic; DNA; DNA biosynthesis; DNA Sequence; drug discovery; Electronics; Enzyme Gene; Funding; Gene-Modified; Genes; genetic variant; Genome; genome-wide; Genomics; Geometry; Goals; Grant; innovation; instrument; instrumentation; Large-Scale Sequencing; Lasers; Light; Methods; Microfluidics; Molecular Genetics; Oligonucleotides; Optics; Phase; plasmid DNA; Polymers; Positioning Attribute; Price; Process; Production; prototype; public health relevance; radio frequency; Reaction; Reading; Research; Scientist; Signal Transduction; Small Business Innovation Research Grant; Solid; Sorting - Cell Movement; Speed (motion); synthetic construct; System; Technology; Time; tool; Vaccine Design; Wor
