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: 7923340
• 负责人: HENDRIK J VILJOEN
• 金额: $ 32万
• 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 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923340
• 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化工程方法，该方法设计了从前体合成5'OH寡核苷酸，长30-40个核苷酸长的前体合成5'OH寡核苷酸的大型DNA分子的过程和平台。该仪器考虑了两种应用：（1）合成寡核苷酸和（2）位置定向诱变的基因组装。该技术的独特功能是组装速度和产品的低误差频率。总体目标（R33的末尾）是开发一种通过键盘与用户相互作用的乐器，并自动执行以下生化步骤：（1）聚合酶链组件（PCA）的1,000-2,000个基本对（BP）双链DNA的DNA碎片（PCA）在比三分之二分钟的情况下，并且在比较少于一个错误的情况下，在两个比较中的每个层中的每一个中，都不是一个错误。 （2）5BRU和6MA HAPEN标记的DNA片段的免疫接触。 （3）用T4聚合酶处理DNA片段。在没有DNTP的情况下，T4聚合酶的3'外丝酶活性从DNA片段的3'末端去除20-30个核苷酸。 （4）按照与连接酶无关的克隆策略，将两个DNA片段结合在一起，其中包含5'单链末端。 R21相结束时的可交付方式将是执行前两个步骤的原型。在此提出的从头基因组装技术允许一旦确定重要的蛋白质，就可以在几个小时的时间范围内合成基因。合成基因可以克隆和过表达，以提供大量的纯蛋白质。纯化的蛋白质可用于生成可用于蛋白质阵列中的高亲和力试剂。蛋白质阵列的开发将提供一个快速有效的程序，以测定正在研究的蛋白质和其他参与毒性反应的蛋白质的表达和调节。快速自动化基因合成​​与蛋白质组学的结合可能会加速新的疾病标志物和药物靶标。

项目成果

Experimental Validation of a Fundamental Model for PCR Efficiency.

• DOI: 10.1016/j.ces.2011.01.029
• 发表时间: 2011-04-15
• 期刊: Chemical engineering science
• 影响因子: 4.7
• 作者: Louw TM;Booth CS;Pienaar E;Termaat JR;Whitney SE;Viljoen HJ
• 通讯作者: Viljoen HJ

Efficiency of the Polymerase Chain Reaction.

• DOI: 10.1016/j.ces.2010.05.046
• 发表时间: 2010-09-01
• 期刊: Chemical engineering science
• 影响因子: 4.7
• 作者: Booth CS;Pienaar E;Termaat JR;Whitney SE;Louw TM;Viljoen HJ
• 通讯作者: Viljoen HJ

Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler.

使用高速热循环仪通过集成聚合酶链组装和 PCR 扩增进行基因合成。

• DOI: 10.1016/j.mimet.2009.09.015
• 发表时间: 2009
• 期刊: Journal of microbiological methods
• 影响因子: 2.2
• 作者: TerMaat,JoelR;Pienaar,Elsje;Whitney,ScottE;Mamedov,TarlanG;Subramanian,Anuradha
• 通讯作者: Subramanian,Anuradha