Ultrafast SBS Method for Large-Scale Human Resequencing

描述（由申请人提供）：识别和理解单核苷酸多态性（snp）的作用将导致遗传疾病状态的准确诊断，确定风险因素，并表征患者的代谢谱。这种技术有望导致预防性治疗，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也太昂贵了。

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.