Real-time DNA Sequencing:Nucleotide Synthesis and Use

描述（申请人摘要）：开发一种 提出了一种对单个DNA分子进行实时测序的方法。工程 聚合酶作为DNA碱基身份的直接分子传感器， 通过合成适当修饰的核苷酸， 最快的酶促DNA测序系统我们的动机 开发单分子测序方法包括消除 目前确定DNA序列所需的中间步骤 信息，从而满足基因组学和医学的需求。 诊断社区我们概述了一系列里程碑， 单分子DNA测序系统的目标。然而，其中只有两个 中期目标是拟议研究的具体目标。在 特别地，我们建议合成适当修饰的核苷酸， 以确定商业上可获得的DNA 聚合酶使用这种修饰的核苷酸来延伸DNA链。 拟定商业应用： 单分子测序系统的开发通常适用于DNA测序。 诊断 此外，样本的并行处理将允许基因组被 序列在一天或更短的时间内，并将是重要的病原体鉴定和 大规模、全面的基因组分析。 该系统消除了排序 反应处理，凝胶或毛细管加载，电泳，有前途的巨大 节省时间、劳动力和每个基地的成本。 这种单分子DNA测序 该系统有望取代目前的DNA测序技术。

DESCRIPTION (Applicant's abstract): A first step toward the development of a method to sequence a single DNA molecule in real-time is proposed. Engineering a polymerase to act as a direct molecular sensor of DNA base identity, coupled with the synthesis of appropriately modified nucleotides, allows us to create the fastest enzymatic DNA sequencing system possible. Our motivation for developing a single-molecule sequencing approach includes eliminating the intermediate steps currently necessary for determining DNA sequence information, thereby meeting the needs of both the genomics and the medical diagnostics communities. We outline a series of milestones towards our ultimate goal of a single-molecule DNA sequencing system. However, only two of these intermediate goals are the specific aims of the proposed research. In particular, we propose to synthesize an appropriately modified nucleotide and to determine the relative efficiency at which commercially available DNA polymerases use this modified nucleotide to extend a DNA strand. PROPOSED COMMERCIAL APPLICATION: Development of a single-molecule sequencing system is generally applicable for DNA diagnostics. Furthermore, parallel processing of samples will allow a genome to be sequence in a day or less, and will be important for pathogen identification and large-scale, comprehensive genome analysis. This system eliminates sequencing reaction processing, gel or capillary loading, and electrophoresis, promising huge savings in time, labor, and cost per base. This single-molecule DNA sequencing system is expected to replace current DNA sequencing technology.