Multiplexed Reactive Sequencing of DNA

DNA 多重反应测序

• 批准号: 6887950
• 负责人: PETER WILLIAMS
• 金额: $ 45.44万
• 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/6887950
• 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。合成的与靶基因上的特定序列互补的DNA引物将被共价拴在玻璃片表面的阵列中的已知位置，并允许与互补的靶基因片段杂交并捕获。在核酸外切酶缺陷的DNA聚合酶存在的情况下，单种荧光标记的脱氧核糖核酸三磷酸(DNTP)将串联询问引物/模板双链。当下一个模板碱基与所提供的dNTP的碱基不互补时，将通过掺入一个或多个脱氧核苷酸单磷酸来延伸其与所提供的dNTP的碱基相邻的模板碱基的那些引物链；当下一个模板碱基不与dNTP互补时，在阵列点中的所有链上的延伸将停止。阵列的定量荧光成像将识别那些延伸的引物并量化所结合的碱基的数量，从而在靶基因上的已知位置读取短长度的序列(一到几个碱基)。然后选择性地破坏荧光标记，并用所有四种类型的dNTP不断重复该循环。在前期工作中开发的手动反应系统将自动用于快速循环。研究的方向将是通过优化连接化学和酶性能，将核苷酸杂质减少到可以忽略的水平，校准荧光反应中的任何上下文相关，以及校正延伸失败产生的信号，从而增加读取长度和序列准确性。最初，每个斑点的读出长度靶至少为50个碱基；沿着靶基因序列，将以较短的间隔(大约相隔20-50个碱基)排列引物，以便能够以短的平行字节快速读出长序列。阵列密度预计为10,000个点，反应周期时间约为2分钟/dNTP，在一张幻灯片上的数据速率约为每分钟1,000个碱基。初步测序研究将针对已知与癌症风险增加相关的基因。随着阅读长度的增加，这项技术将被应用于从头测序，方法是发现与载体序列对应的通用引物上的克隆模板。