Multiplexed Reactive Sequencing of DNA

描述（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特定序列互补的DNA引物将共价系在玻璃载玻片表面阵列的已知位置，并允许与互补的目标基因片段杂交并捕获。在核酸外切酶缺陷DNA聚合酶存在的情况下，引物/模板双链将被单种荧光标记的脱氧核糖核苷酸三磷酸（dNTP）连续询问。引物3′端附近的模板碱基与提供的dNTP碱基互补的引物链将通过加入一个或几个脱氧核苷酸单磷酸而延长；当下一个模板基与dNTP不互补时，扩展将在数组点的所有链上停止。该阵列的定量荧光成像将识别那些已经扩展的引物，并量化纳入的碱基数量，从而读取目标基因上已知位置的短序列（一个到几个碱基）。然后有选择地破坏荧光标记，循环不断重复所有四种类型的dNTP。在初步工作中开发的手动反应系统将自动进行快速循环。研究将通过优化附着化学和酶的性能，将核苷酸杂质减少到可以忽略不计的水平，校准荧光响应中的任何上下文依赖，以及纠正由延伸失败引起的信号，来增加读取长度和序列准确性。最初，每个位点的读取长度目标至少为50个碱基；引物将沿着目标基因序列以短间隔（约20-50个碱基间隔）排列，以便长序列可以在短并行字节中快速读出。预计阵列密度为10万个点，反应周期时间约为2分钟/dNTP，单片上的数据速率约为每分钟1000个碱基。初步的测序研究将针对已知与癌症风险升高相关的基因。随着读取长度的增加，该技术将应用于从头测序，通过定位克隆模板退火到与载体序列对应的通用引物。

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.