Multiplexed Reactive Sequencing of DNA

DNA 多重反应测序

• 批准号: 6953769
• 负责人: PETER WILLIAMS
• 金额: $ 49万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6953769
• 关键词: DNA directed DNA polymerase; cancer risk; charge coupled device camera; chromosomes; computer program /software; computer system design /evaluation; deoxyribonucleoside triphosphate; method development; microarray technology; nucleic acid hybridization; nucleic acid sequence; polymerase chain reaction; reagent /indicator

DESCRIPTION (provided by applicant): A new approach to DNA sequencing is proposed. DNA primers synthesized to be complementary to specific sequences on targeted genes will be covalently tethered in known locations in an array on the surface of a glass slide and allowed to hybridize with and capture complementary target gene fragments. The primer/template duplexes will be serially interrogated by single species of fluorescently-labeled deoxyribonucleotide triphosphate (dNTP) in the presence of an exonuclease-deficient DNA polymerase. Those primer strands for which the template base adjacent to the 3' end of the primer is complementary to the base of the dNTP supplied will be extended by incorporation of one or several deoxynucleotide monophosphates; extension will cease, on all strands in an array spot, when the next template base is not complementary to the dNTP. Quantitative fluorescent imaging of the array will identify those primers that have extended and quantify the number of bases incorporated, thus reading a short length of sequence (one to several bases) at a known location on the target gene. The fluorescent label is then selectively destroyed and the cycle is continually repeated with all four types of dNTP. A manual reaction system developed in preliminary work will be automated for rapid cycling. Studies will be directed towards increasing read length and sequence accuracy by optimizing attachment chemistries and enzyme performance, reducing nucleotide impurities to negligible levels, calibrating any context-dependent in the fluorescence response, and correcting for signals arising from extension failure. Initially, the read length target per spot is at least 50 bases; primers will be spaced at short intervals (about 20-50 bases apart) along the target gene sequences so that a long sequence can rapidly be read out in short parallel bytes. Array densities > 10,000 spots are anticipated, with reaction cycle times approximately 2 min/dNTP giving data rates about 1,000 bases per minute on a single slide. Initial sequencing studies will address genes known to be associated with an elevated risk of cancer. As read length is increased, the technology will be applied to de novo sequencing by spotting cloned templates annealed to a universal primer corresponding to the vector sequence.

描述（由申请人提供）：提出了一种新的 DNA 测序方法。合成的与目标基因上的特定序列互补的 DNA 引物将共价连接在载玻片表面阵列中的已知位置，并允许与互补的目标基因片段杂交并捕获互补的目标基因片段。在存在核酸外切酶缺陷的 DNA 聚合酶的情况下，引物/模板双链体将被单一种类的荧光标记的脱氧核糖核苷酸三磷酸 (dNTP) 连续询问。与引物3'端相邻的模板碱基与所提供的dNTP的碱基互补的那些引物链将通过掺入一个或多个脱氧核苷酸单磷酸而延伸；当下一个模板碱基与 dNTP 不互补时，阵列点中所有链上的延伸将停止。阵列的定量荧光成像将识别那些已延伸的引物并量化掺入的碱基数量，从而读取目标基因上已知位置处的短长度序列（一个到几个碱基）。然后荧光标记被选择性破坏，并且使用所有四种类型的 dNTP 不断重复该循环。前期工作中开发的手动反应系统将实现自动化以实现快速循环。研究将致力于通过优化附着化学和酶性能、将核苷酸杂质减少到可忽略的水平、校准荧光响应中的任何背景依赖性以及校正延伸失败产生的信号来增加读取长度和序列准确性。最初，每个点的读长目标至少为 50 个碱基；引物将沿着目标基因序列以短间隔（大约相隔20-50个碱基）间隔开，以便可以以短的并行字节快速读出长序列。预计阵列密度 > 10,000 个点，反应周期时间约为 2 分钟/dNTP，单张载玻片上的数据速率约为每分钟 1,000 个碱基。最初的测序研究将针对已知与癌症风险升高相关的基因。随着读长的增加，该技术将应用于从头测序，通过点样克隆模板与对应于载体序列的通用引物退火。