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：
10552680
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/10552680
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 Ligation DNA biosynthesis Derivation procedure Device Designs Dimensions Engineering Fluorescence Genome Genomic DNA Genomics Glass Grain Health Human Human Genome Image Image Analysis In Vitro Ionic Strengths Laboratories Length Ligation Maps Measurement Mediating Methods Microscopy Modality Modeling Modification Molecular Molecular Conformation Names Nucleic Acids Oligonucleotides Optics Pathway interactions Peptides Performance Periodicity Phase Physical condensation Polyamines Process Reagent Reproducibility Research Scheme Solid Stretching Surface System Techniques Technology Validation copolymer design electric field fabrication foot manufacture microscopic imaging molecular modeling monomer novel operation physical property polyacrylamide polyacrylamide hydrogels research and development response single molecule synergism 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，长期目标是开发系统的系统直接，无细胞，经过验证的非常大的DNA组件的制造，用于合成的应用应用和基因组组件。该系统 - “ Gensyn” - 将利用大型DNA以综合支持，以补充和协同基于细胞的方式非常大的DNA分子的系列组装技术。拟议的研究是基于i）支持化学和酶促过程的结合方法； ii）无剪切流体操作； iii）增强杂交/连接产量和iv）验证的新方法。这些设备专为支持组装和图像分析的温和流体操作而设计大型DNA构建体通过落叶显微镜。 Gensyn周期的第一步将结合DNA分子与与结合的寡核苷酸杂交的支持衍生化的玻璃表面。杂交和连接步骤将使用物理优化和化学操作。修改的大型DNA将用作耦合器分子，支持 Gensyn周期。总体周期性能指标（耦合产量随循环的函数将评估数量，分子断裂，试剂保留，时机和酶活性）以计算机模型为指导和指导的方式。这项研究将有助于进步基因组和人类健康的基本研究。