A Rational Design of a Platform for de novo Gene Synthesis

描述(由申请人提供)： 本项目描述了一种生物化学工程方法，以设计一种由前体合成的30-40个核苷酸的5‘-羟基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了两个应用：(1)从合成的寡核苷酸中组装基因和(2)定点突变。该技术的独特之处在于组装速度快，产品出错率低。总的目标(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)聚合链组装(PCA)在两个试管中的每个试管中在不到30分钟内将1,000-2,000个碱基对(BP)双链DNA片段组装在一起，并且准确度低于1,000个误差/1,000个碱基对。(2)免疫捕获5BrU和6 mA半抗原标记的DNA片段。(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‘外切酶活性从DNA片段的3’端去除20-30个核苷酸。(4)按照不依赖连接酶的克隆策略，将含有5‘端单链末端的两个DNA片段连接在一起。R21阶段结束时的可交付成果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在几个小时的时间尺度上合成基因，一旦确定了重要的蛋白质。合成的基因可以被克隆和过度表达，以提供大量的纯蛋白。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。蛋白质阵列的发展将提供一种快速而有效的程序来分析被研究蛋白质和其他参与毒性反应的蛋白质的表达和调控。快速自动基因合成与蛋白质组学的结合可能会加速新的疾病标记物和药物靶点的开发。

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.