Multiplexed Reactive Sequencing of DNA

描述（由申请人提供）：提出了一种新的DNA测序方法。被合成为与靶基因上的特定序列互补的DNA引物将被共价连接在载玻片表面上的阵列中的已知位置，并允许与互补靶基因片段杂交并捕获互补靶基因片段。在存在核酸外切酶缺陷型DNA聚合酶的情况下，通过单一种类的荧光标记脱氧核糖核苷酸三磷酸（dNTP）对引物/模板双链体进行连续询问。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几个脱氧核苷酸单磷酸来延伸;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鉴定已经延伸的那些引物并定量掺入的碱基的数量，从而阅读靶基因上已知位置处的短长度序列（一个至几个碱基）。然后选择性地破坏荧光标记，并使用所有四种类型的dNTP不断重复该循环。在初步工作中开发的手动反应系统将自动化，以实现快速循环。研究将通过优化连接化学和酶性能、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上下文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度。最初，每个斑点的读取长度靶标为至少50个碱基;引物将沿着靶基因序列以短间隔（约20-50个碱基间隔）隔开，使得长序列可以以短的平行字节快速读出。预期阵列密度> 10，000个点，反应循环时间约为2分钟/dNTP，在单个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。随着读取长度的增加，该技术将通过点样与对应于载体序列的通用引物退火的克隆模板而应用于从头测序。

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.