DNA polymerase for single molecule DNA sequencing

用于单分子 DNA 测序的 DNA 聚合酶

• 批准号: 6536483
• 负责人: JOHN G. K. WILLIAMS
• 金额: $ 50.68万
• 依托单位: LI-COR BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2003-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6536483
• 关键词: DNA directed DNA polymerase; DNA footprinting; bacteriophage T7; biochemical evolution; biotechnology; combinatorial chemistry; fluorescent dye /probe; gene mutation; high throughput technology; microarray technology; molecular cloning; nanotechnology; nucleic acid sequence; nucleotide analog; polymerase chain reaction; protein engineering; protein purification; recombinant proteins; site directed mutagenesis; technology /technique development

DESCRIPTION (provided by applicant): We are developing a new technology for single molecule DNA sequencing based on the incorporation of dNTPs labeled on the g-phosphate with fluorescent dyes. The label is cleaved from the nucleotide along with pyrophosphate when the nucleotide is incorporated into DNA by a polymerase. The released label is sorted from intact g-dNTPs in a microfluidics system and is identified in real time by single molecule fluorescence detection optics. In this proposal, we develop a novel nucleotide chemistry to enable the sorting scheme and we use directed evolution methods to adapt polymerases for utilization of the g-dNTPs. Single molecule sequencing promises to reduce the cost of DNA sequencing by several orders of magnitude compared to current methods. PROPOSED COMMERCIAL APPLICATIONS: A genomic DNA sequencing technology that reduces cost and eliminates the need for cloning, physical mapping and electrophoresis would have great appeal to both the academic and industrial genomic research communities.

描述（由申请人提供）：我们正在开发一项新技术 基于掺入标记的 dNTP 的单分子 DNA 测序 g-磷酸盐与荧光染料。标签从核苷酸上切下 当核苷酸被掺入 DNA 时，与焦磷酸一起 聚合酶。释放的标签是在微流体中从完整的 g-dNTP 中分选出来的 系统并通过单分子荧光检测实时识别 光学。在本提案中，我们开发了一种新型核苷酸化学，使 排序方案，我们使用定向进化方法来适应聚合酶 g-dNTP 的利用。单分子测序有望减少 DNA测序的成本比目前高出几个数量级 方法。 拟议的商业应用： 基因组 DNA 测序技术可以降低成本并消除克隆、物理作图和电泳的需要，这对学术和工业基因组研究界都具有巨大的吸引力。