GenSyn: A Cycle for Serial Assembly of Very Large DNA Molecules

点击翻译按钮获取中文摘要

Abstract The broad, long-term objectives of the proposed research are development of a system for direct, cell-free, validated fabrication of very large DNA assemblies, for applications in synthetic applications and genome assembly. This system – “GenSyn” – will harness the advantages of large DNAs for synthesis on supports in ways that will complement and synergize cell-based techniques for the serial assembly of very large DNA molecules. The proposed research is based on i) binding approaches supporting chemical and enzymatic processes; ii) shear-free fluidic operations; iii) new methods for enhancing hybridization/ligation yields and iv) validations. The devices are designed for gentle fluidic operations supporting assembly and image analysis of large DNA constructs by epifluorescence microscopy. The first step in the GenSyn cycle will be binding DNA molecules to a support by hybridization with oligonucleotides that are bound to a derivatized glass surface. The hybridization and ligation steps will be optimized using physical and chemical manipulations. Modified large DNAs will be used as coupler molecules, supporting GenSyn cycles. Overall cycle performance metrics (coupling yields as a function of cycle number, molecule breakage, reagent retention, timings and enzymatic activity) will be assessed in ways that will inform and be guided by computer models. This research will help advance fundamental studies on genomes and human health.