Genome-scale De Novo DNA Synthesis on Electronic p-Chips with Sorting

 描述（由申请人提供）：本项目的目的是开发和验证一种合成大量DNA片段寡脱氧核苷酸的新方法。该方法被称为“序列指导的DNA合成”，涉及使用小型电子电路“p芯片”，每个芯片在用激光照射时通过RF传输唯一的ID号。在大规模平行形式中，使用p-Chips作为固相合成寡核苷酸。p-Chip ID提供了在合成进行时将给定DNA序列与该特定p-Chip相关联的能力。该系统的关键部分是一个高速仪器，即分选机，它读取每个p-Chip的ID，然后根据p-Chip ID将p-Chip分选到适当的反应容器中，从而确定特定的DNA序列。该系统完成后，将能够在很短的时间内生成合成DNA片段，成本远低于目前的方法。该项目的工作计划是在第一阶段初步证明在目前可用的0.5 mm p芯片上合成DNA的能力，并显示分选的可行性。在此之后，在第二阶段，将转移到一个新的p芯片设计，其中每个是千分之一的0.5毫米p芯片的体积。主要目标将是为这些超小芯片构建分类器，并制定大规模序列指导组合DNA合成的工作方法。我们还将通过在p-Chips上合成大量寡核苷酸并创建10 kb长的基因片段来证明该方法的适用性。我们将获得克隆时的DNA序列错误率，并确定基因组装和构建长片段染色体的最佳方法。以低廉的价格快速生产DNA的前景在与合成基因组学相关的许多领域具有深远的影响，这些领域需要新的工具。这包括酶、能源生产、疫苗设计和许多其他应用的修饰基因的设计。

 DESCRIPTION (provided by applicant): The purpose of the present project is to develop and validate a new method for synthesizing large number of DNA fragments, oligodeoxynucleotides. The method, which is called "sequence-directed DNA synthesis", involves the use of small electronic circuits, "p-Chips", each of which transmits a unique ID number via RF when illuminated with laser light. In a massively parallel format, oligonucleotides are synthesized using p-Chips as the solid phase. The p-Chip ID provides the ability to associate a given DNA sequence with that particular p-Chip as synthesis proceeds. A key part of the system is a high-speed instrument, the sorter, that reads the ID of each p-Chip and then sorts p-Chips into the appropriate reaction vessel based on the p-Chip ID, thus the specific DNA sequence. The system, when completed, will be able to generate synthetic DNA fragments in very short times and at costs that are much lower than current methods. The work plan for the project is to initially, in Phase I, demonstrate the ability to synthesize DNA on currently available 0.5 mm p-Chips and to show feasibility of sorting. Following this, in Phase II, will shift to a new p-Chip design, each of which is one thousandth the volume of the 0.5 mm p-Chip. The main goals will be to build the sorter for these ultra-small chips, and to formulate a working method for large-scale sequence-directed combinatorial DNA synthesis. We will also demonstrate applicability of the method by synthesizing a large number of oligonucleotides on p-Chips and creating a 10 kb long gene fragment. We will obtain the DNA sequence error rate upon cloning and identify the best methods for gene assembly and construction of long segments of chromosomes. The prospect of rapidly producing DNA at low prices has profound implications in a number of fields related to synthetic genomics where new tools are needed. This includes the design of modified genes for enzymes, energy production, vaccine designs and many other applications.