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

摘要 拟议研究的广泛和长期目标是开发一个系统，用于 直接、无细胞、经过验证的超大型DNA组件的制造，用于合成 应用和基因组组装。这个名为“GenSyn”的系统将利用 用于在载体上合成的大DNA，以补充和协同细胞为基础的方式 超大DNA分子的串联组装技术。拟议的研究是 基于i)支持化学和酶过程的结合方法；ii)无剪切 流体操作；iii)提高杂交/连接产量的新方法和iv)验证。 这些设备专为温和的流体操作而设计，支持组装和图像分析 通过荧光显微镜观察到大的DNA结构。GenSyn周期的第一步将是 通过与结合到载体上的寡核苷酸杂交将DNA分子结合到载体上 衍生的玻璃表面。杂交和连接步骤将使用物理优化 和化学操作。修饰的大DNA将被用作偶联剂分子，支持 GenSyn周期。整体循环性能指标(耦合收益率作为循环的函数 数量、分子断裂、试剂保留、计时和酶活性)将被评估 以提供信息并由计算机模型指导的方式。这项研究将有助于推动 基因组与人类健康的基础研究。

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.