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

GenSyn：非常大的 DNA 分子的串行组装循环

基本信息

批准号：
10369379
负责人：
DAVID C. SCHWARTZ
金额：
$ 26.55万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-19 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10369379
关键词：
Adsorption Attenuated Bacterial Artificial Chromosomes Binding Biological Assay Buffers Cells Charge Chemicals Chromosomes Complement Computer Models Computer Simulation Coupling DNA DNA Binding DNA biosynthesis Device Designs Diffuse Dimensions Engineering Fluorescence Genome Genomic DNA Genomics Glass Grain Health Human Human Genome Hybrids Image Image Analysis In Vitro Ionic Strengths Laboratories Length Ligation Liquid substance Measurement Mediating Methods Microscopy Modality Modeling Modification Molecular Molecular Conformation Names Oligonucleotides Optics Pathway interactions Peptide Nucleic Acids Performance Periodicity Phase Physical condensation Polyamines Process Reagent Reproducibility Research Scheme Seeds Solid Stretching Surface System Techniques Technology Validation base design electric field foot microscopic imaging molecular modeling monomer novel operation physical property polyacrylamide polyacrylamide hydrogels research and development response single molecule synthetic construct synthetic nucleic acid technology development

项目摘要

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.

抽象的拟议研究的广泛、长期目标是开发一个系统直接、无细胞、经过验证的非常大的 DNA 组装体的制造，用于合成应用该系统——“GenSyn”——将利用以下优势：用于在支持物上合成的大DNA，以补充和协同基于细胞的方式所提议的研究是用于连续组装非常大的 DNA 分子的技术。基于 i) 支持化学和酶促过程的结合方法；ii) 无剪切流体操作；iii) 提高杂交/连接产量的新方法；iv) 验证。该设备专为温和的流体操作而设计，支持装配和图像分析 GenSyn 循环的第一步将是通过落射荧光显微镜分析大型 DNA 构建体。通过与结合的寡核苷酸杂交将DNA分子结合到支持物上衍生化的玻璃表面将使用物理方法进行优化。修饰的大DNA将用作偶联分子，支持 GenSyn 循环。总体循环性能指标（耦合产量作为循环的函数）数量、分子断裂、试剂保留、时间和酶活性）将被评估这项研究将有助于推动计算机模型的发展。基因组和人类健康的基础研究。