High fidelity low cost manufacture of synthetic DNA

合成 DNA 的高保真低成本制造

• 批准号: 7052331
• 负责人: NICOLAY KULIKOV
• 金额: $ 36.22万
• 依托单位: DNA TWOPOINTO, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-14 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7052331
• 关键词: deoxyribopolynucleotide; genes; genetic manipulation; nucleic acid chemical synthesis; oligonucleotides

DESCRIPTION (provided by applicant): The long term objective of this project is to develop technology to provide low-cost (<$0.50/bp), rapid synthesis of complete genes (500 bp to 25 kb). Rapid, accurate and affordable gene synthesis will provide a direct link between genomic information and hypothesis-driven experimental science, improving our ability to understand disease processes and design therapeutic agents. Examples of the ways in which gene synthesis can impact health-related projects include codon optimization, splice variant construction and protein engineering for expression of human and pathogen proteins in heterologous hosts for structural genomics, antigen production and use as targets / reporters for drug discovery efforts. Gene synthesis is currently too slow and expensive to meet the needs of the genomic and health sciences community. Current prices are around $2/bp with turnaround times of 3-6 weeks for a gene of 1 kb. The major factors governing price are the costs of building block synthesis and sequence confirmation. The specific aim of this project is to develop methods and machines to synthesize 1-2 pmol of oligonucleotides with isolated purity > 90% and to reliably assemble these into genes. We aim to lower the cost of building block synthesis 3- to 10-fold by combining advanced chemistry with alternative engineering allowing a reduction in reaction volumes and reagent concentrations. It is essential for this application that this reduced cost is accompanied by an increase in quality: lower quality oligonucleotides assemble into genes that have increased error frequencies and thus require additional downstream manipulations and sequence verification, increasing the overall cost of production. We will therefore optimize synthesis on a 2- dimensional support to improve the stepwise coupling efficiency to >99.5%; in Phase I we demonstrated the feasibility of this by obtaining coupling efficiencies of 99.9% using modified chemistries and procedures. Small scale, low cost, high coupling efficiency oligonucleotide synthesis will allow greater flexibility in oligonucleotide gene building block design, remove the need for oligonucleotide purification prior to assembly into complete genes, reduce sequence confirmation costs and shorten order to delivery times. To achieve this, in Phase II we will develop an automated 96 channel instrument for synthesizing oligonucleotides with >90% purity. The machine will be integrated into our existing high throughput gene production line at DNA2.0. A low cost high fidelity oligonucleotide manufacturing process may have additional applications in related areas including diagnostic microarrays, real time PCR and RNAi-reagents.

描述（由申请人提供）：该项目的长期目标是开发技术以提供低成本（<$ 0.50/bp），快速合成完整基因（500 bp至25 kb）。快速，准确和负担得起的基因合成将提供基因组信息与假设驱动的实验科学之间的直接联系，从而提高了我们了解疾病过程和设计治疗剂的能力。基因合成影响与健康相关项目的方式的例子包括密码子优化，剪接变异构建和蛋白质工程，以表达人和病原体蛋白在异源宿主中用于结构基因组学，抗原产生以及用作药物发现工作的靶标 /记者。基因合成目前太慢且昂贵，无法满足基因组和健康科学社区的需求。目前的价格约为2美元/bp，基因为1 kb的周转时间为3-6周。关于价格的主要因素是构建基块合成和序列确认的成本。该项目的具体目的是开发方法和机器，以隔离纯度> 90％合成1-2 pmol的寡核苷酸，并可靠地将其组装成基因。我们的目标是通过将晚期化学与替代工程相结合，允许降低反应量和试剂浓度的替代工程，以降低3至10倍的构建块合成成本。对于此应用，这种降低的成本伴随着质量的提高至关重要：降低质量的寡核苷酸将误差频率增加，因此需要额外的下游操作和序列验证，从而增加了总体生产成本。因此，我们将优化在2维支持上的合成，以提高逐步耦合效率> 99.5％；在第一阶段，我们通过使用改良的化学和程序获得99.9％的耦合效率来证明了这一点的可行性。小规模，低成本，高耦合效率寡核苷酸的合成将使寡核苷酸基因构建块设计更大的灵活性，消除在组装成完整基因之前对寡核苷酸纯化的需求，减少序列确认成本并短短订购序列。为了实现这一目标，在第二阶段，我们将开发一种自动化的96通道仪器，用于合成纯度> 90％的寡核苷酸。该机器将在DNA2.0的现有高通量基因生产线中集成到我们现有的高通量基因生产线中。低成本的高富达寡核苷酸制造过程可能在相关领域还有其他应用，包括诊断微阵列，实时PCR和RNAi-REAGENTS。