Ultrafast SBS Method for Large-Scale Human Resequencing

用于大规模人体重测序的超快 SBS 方法

• 批准号: 6953265
• 负责人: Michael L. Metzker
• 金额: $ 54.96万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-29 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6953265
• 关键词: DNA replication; chemical synthesis; fluid flow; high throughput technology; microarray technology; molecular biology; nucleic acid sequence; nucleoside triphosphate; single nucleotide polymorphism; technology /technique development

DESCRIPTION (provided by applicant): Identifying and understanding roles of single nucleotide polymorphisms (SNPs) will lead to accurate diagnosis of inherited disease states, determination of risk factors, and characterization of patients' metabolic profiles. Such technology promises to lead to prophylactic treatments to delay the onset or progression of disease, and prescriptions of the safest and most efficacious medications. Current DNA sequencing technology, however, is too slow and expensive for these tasks. Here, we propose to develop an ultrafast DNA sequencing system featuring sequencing-by-synthesis (SBS) on high-density oligonucleotide arrays, each with approximately one million primer features. The collaborative team involved in this project was responsible for some of the earliest published work on SBS, and recognize the fundamental challenge that any method based on this approach must address before tangible progress to a practical system can be made. That is, to identify combinations of appropriately modified nucleoside triphosphates that will be accepted, efficiently and with high fidelity, by suitably mutated DNA replicating enzymes. Consequently, this proposal features a strong synthetic chemistry component featuring two laboratories focused on the preparing nucleoside triphosphates with fluorescent, labile 3'-protecting groups. It also describes molecular biology to produce relatively large libraries of mutated polymerases. Even though the numbers of modified enzymes generated is high, the mutations will focus on key structural regions to maximize the chances of finding suitable systems. This molecular biology component is coupled with a combinatorial screen to rapidly identify suitable enzyme/modified dNTP pairs. Once suitable combinations are identified, then the SBS methodology will be implemented using high-density arrays that, uniquely, orientate oligonucleotides in the desired 5'-> 3' direction. This core technology fits into a broader, comprehensive research plan encompassing microfluidics for sample manipulation and delivery of the DNA to the SBS system, fluorescent imaging via our proprietary Pulse-Multiline Excitation (PME) system, computational methods for identifying an optimal tiling path and thermodynamic properties of oligonucleotides across whole chromosomes, and informatics to process and store the data generated. The overall goal is, by the end of year three, to complete sequencing of chromosomes 3, 12 & X, which cover approximately 0.5 gigabases and would lay the foundation for whole genome sequencing.

描述（由申请人提供）：识别和理解单核苷酸多态性（SNP）的作用将有助于准确诊断遗传性疾病状态、确定危险因素以及表征患者的代谢特征。此类技术有望带来预防性治疗，以延缓疾病的发作或进展，并开出最安全、最有效的药物处方。然而，目前的 DNA 测序技术对于这些任务来说太慢且昂贵。 在这里，我们建议开发一种超快 DNA 测序系统，其特点是在高密度寡核苷酸阵列上进行合成测序 (SBS)，每个阵列具有大约一百万个引物特征。参与该项目的协作团队负责 SBS 上一些最早发表的工作，并认识到任何基于此方法的方法都必须解决基本挑战，然后才能在实际系统方面取得切实进展。也就是说，鉴定适当修饰的三磷酸核苷的组合，这些组合将被适当突变的DNA复制酶有效且高保真地接受。因此，该提案具有强大的合成化学成分，其中两个实验室专注于制备具有荧光、不稳定 3'-保护基团的核苷三磷酸。它还描述了产生相对较大的突变聚合酶文库的分子生物学。尽管生成的修饰酶的数量很高，但突变将集中在关键结构区域，以最大限度地提高找到合适系统的机会。该分子生物学组件与组合筛选相结合，可快速识别合适的酶/修饰的 dNTP 对。一旦确定了合适的组合，则将使用高密度阵列实施 SBS 方法，该阵列独特地将寡核苷酸定向在所需的 5'-> 3' 方向。这项核心技术适合更广泛、全面的研究计划，包括用于样品操作和将 DNA 输送到 SBS 系统的微流体、通过我们专有的脉冲多线激发 (PME) 系统进行的荧光成像、用于识别最佳平铺路径和热力学的计算方法整个染色体上寡核苷酸的特性，以及处理和存储生成的数据的信息学。总体目标是，到第三年年底，完成 3、12 和 X 号染色体的测序，覆盖约 0.5 个千兆碱基，将为全基因组测序奠定基础。