Research Project
8780542
DESCRIPTION (provided by applicant): Over the last 3 decades the cost of synthetic DNA for routine applications such as DNA sequencing, PCR and hybridization analysis has become a substantial part of many laboratory budgets. Additionally, length and quality issues limit the expansion of numerous biological applications which could impact human health developments. Finally, current DNA synthesis methods, developed 30 years ago are organic chemistry based and produce toxic waste mixtures that are difficult and costly to dispose of. This proposal describes a novel method that will lead to reduced costs in every molecular biology laboratory and enable the development of new synthetic biology applications such as faster development of vaccines, biomolecular computation, reprograming of cells and improved cellular therapeutics. The resulting massively parallel synthesis capability developed in Phase II of this project will be put to use as a custom synthesis service, similar to how custom oligos are ordered, produced and delivered today, run by Molecular Assembly. The enzyme terminal deoxynucleotidyl transferase acts by adding one base at a time to a single stranded DNA in a template-independent fashion. The goal of this project is to harness the remarkable properties of TdT which will polymerize homopolymers thousands of nucleotides long, to instead produce sequence specific polynucleotides of similar lengths. The approach describes development of two deoxyribonucleotide triphosphate (dNTP) analogs that are modified in such a way so that they are a) compatible with TdT and are readily added to a strand of DNA being synthesized, b) blocked in such a way that leads to the addition of one and only one nucleotide of choice at a time and c) after being added to the growing strand, able to be de- blocked in such a way that regenerates a natural DNA strand. Phase I covers the development of such dNTP analogs and their use to prove the ability to make a short sequence specific nucleic acid. Phase II will lead t optimization of all four dNTP analogs, cycle conditions, automation and the demonstration of the full capabilities of this novel, synthetic approach for polydeoxynucleotides. In 1981 it would have been difficult to envision the specific fundamental roles DNA synthesis would eventually play in modern biology; primers for sequencing the human genome, PCR and hybridization arrays. One can only imagine how on-demand, high purity, low cost polynucleotides will enable a new era of biological & clinical applications.
