A Rational Design of a Platform for de novo Gene Synthesis

基因从头合成平台的合理设计

• 批准号: 7681570
• 负责人: HENDRIK J VILJOEN
• 金额: $ 32.33万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7681570
• 关键词: Affinity; Ampicillin Resistance; Base Pairing; Biochemical; Biochemical Reaction; Biochemistry; Biological Assay; Cells; Chemistry; Chimeric Proteins; Chinese Hamster Ovary Cell; Cloning; DNA; DNA Structure; Development; Devices; Diagnostic; Digestion; Disease Marker; Drug Delivery Systems; Electronics; Engineering; Exonuclease; Frequencies; Gases; Genes; Goals; Haptens; Hour; Human; Immunoglobulin G; Investigation; Label; Lactamase; Length; Ligase; Manuals; Mind; Nucleotides; Oligonucleotides; Phase; Plasmids; Polymerase; Procedures; Process; Protein Array; Protein C; Proteins; Proteomics; Protocols documentation; Reaction; Reagent; Regulation; Replication Origin; Reporting; Research; Sampling; Site-Directed Mutagenesis; Speed; Staging; Structure; Synthetic Genes; System; Technology; Thrombomodulin; Time; base; design; ds-DNA; gene synthesis; instrument; mathematical model; mitochondrial genome; mutant; overexpression; phosphoramidite; programs; prototype; receptor; research study; response; sensor; spleen exonuclease; synthetic construct; time use

DESCRIPTION (provided by applicant): This project describes a biochemical engineering approach to design a process and platform for the de novo synthesis of large DNA molecules from precursor synthetic 5'OH oligonucleotides, 30-40 nucleotides long. The instrument is engineered with two applications in mind: (1) assembly of genes from synthetic oligonucleotides and (2) site-directed mutagenesis. The unique features of the technology are the speed of assembly and the low error frequencies of products. The overall objective (end of R33) is to develop an instrument that interacts with users by keyboard and performs the following biochemical steps automatically: (1) Polymerase chain assembly (PCA) of 1,000-2,000 base pairs (bp) double-stranded DNA fragments in each of two cuvettes in less than thirty minutes and with an accuracy of less than one error/1,000 bp. (2) Immunocapture of the 5BrU and 6mA hapten-labeled DNA fragments. (3) Treat the DNA fragments with T4 polymerase. In the absence of dNTP's, the 3' exonuclease activity of the T4 polymerase removes 20-30 nucleotides from the 3' ends of the DNA fragments. (4) Join the two DNA fragments, which contain 5' single stranded ends, following a ligase-independent cloning strategy. The deliverable at the end of the R21 phase would be a prototype that performs the first two steps. The de novo gene assembly technology proposed here allows genes to be synthesized on a time scale of a few hours once important proteins are identified. The synthetic genes can be cloned and overexpressed to provide a large quantity of pure protein. Purified proteins may be used to generate high-affinity reagents that can be employed in protein arrays. The development of protein arrays will provide a quick and efficient procedure to assay the expression and regulation of the protein under investigation and of other proteins involved in toxic-response. The combination of rapid automated gene synthesis with proteomics may accelerate the development of new disease markers and drug targets.

描述（由申请人提供）： 该项目描述了一种生物化学工程方法，用于设计从前体合成的30-40个核苷酸长的5 'OH寡核苷酸从头合成大DNA分子的过程和平台。该仪器的设计考虑到两个应用：（1）从合成寡核苷酸组装基因和（2）定点诱变。该技术的独特之处在于装配速度快和产品错误频率低。总体目标（R33结束）是开发一种通过键盘与用户交互并自动执行以下生化步骤的仪器：（1）在两个比色皿中的每一个中在不到30分钟内聚合酶链组装（PCA）1，000 - 2，000个碱基对（bp）的双链DNA片段，准确度小于1个错误/1，000 bp。(2)5 BrU和6 mA半抗原标记的DNA片段的免疫捕获。(3)用T4聚合酶处理DNA片段。在不存在dNTP的情况下，T4聚合酶的3'核酸外切酶活性从DNA片段的3'末端去除20-30个核苷酸。(4)按照连接酶非依赖性克隆策略，连接两个含有5'单链末端的DNA片段。R21阶段结束时的交付物将是执行前两个步骤的原型。这里提出的从头基因组装技术允许基因在几个小时的时间尺度上合成，一旦重要的蛋白质被识别。合成基因可以被克隆和过表达以提供大量的纯蛋白质。纯化的蛋白质可用于产生可用于蛋白质阵列的高亲和力试剂。蛋白质芯片技术的发展将为研究受试蛋白质以及其他参与毒性反应的蛋白质的表达和调控提供一种快速有效的方法。快速自动化基因合成与蛋白质组学的结合可能会加速新疾病标志物和药物靶点的开发。