DNA SEQUENCING--ITERATIVE STEPS BY PRODUCT REGENERATION

描述（改编自研究者摘要）：在此授予 应用程序，研究人员建议开发一种方法，可以测序 许多DNA片段平行，而没有DNA片段的凝胶解析。 DNA 测序目前使用桑格法，需要凝胶解析步骤 这限制了输出和准确性。 通过杂交进行测序受到以下因素的阻碍： 模板中的反向重复序列和序列组成对双链体的影响 阵 这里提出的方法对几乎完全是 双链，防止DNA二级结构导致的序列错误 结构. 第一个具体目标是开发一个协议， 在许多载体插入物的每一个中平行地测序150个核苷酸。 这 该方法使用连接编码识别结构域的接头， IIS类限制性内切酶，并含有各自独特的序列 测序周期 PCR无缝附加限制性内切酶 识别结构域，并用IIS类限制性内切酶消化 识别附加的识别结构域的DNA片段， 产生由短突出端组成的DNA模板。 测序 突出端模板中的核苷酸通过模板指导的 通过与标记的ddNTP聚合或通过模板定向连接， 标签适配器 每个循环所特有的连接接头序列 允许PCR步骤仅以指数方式再生所需的模板 在每次迭代期间的前体;因此，该授权测试假设， 体外指数DNA扩增可以克服背景信号 这种累积使替代的迭代DNA测序方法失效。 此外，DNA测序发生在跨越到核苷酸的数量 分离识别和切割结构域，最大化DNA的跨度 对给定数量的核苷酸进行测序。 第二个具体目标是 开发一个协议来阻止预先存在的内部类IIS识别 被测序的DNA片段中的结构域。 利用现有文书和 微量滴定板，该方法将能够测序大于1000 同时插入载体，阅读大于100，000个核苷酸。 在 将来这种方法可以适用于生物芯片形式。 工作 在这个补助金申请中提出的将允许至少150个测序 在DNA文库中的数千个插入物中的每一个中的核苷酸。 测序的 段将是正确的，并有足够的长度，广泛的自下而上 基因组序列重建

DESCRIPTION (Adapted from the Investigator's Abstract): In this grant application, the investigator proposes to develop a method that can sequence many DNA fragments in parallel without gel resolution of DNA fragments. DNA sequencing currently uses the Sanger method, requiring a gel resolution step that limits output and accuracy. Sequencing by hybridization is hindered by inverted repeats in the template and sequence composition effects on duplex formation. The method presented here sequences DNA that is almost entirely double-stranded, preventing sequence errors resulting from DNA secondary structures. The first specific aim is to develop a protocol that can sequence 150 nucleotides in each of many vector inserts in parallel. This method uses ligation of an adaptor that encodes the recognition domain for a class-IIS restriction endonuclease and contains a sequence unique to each sequencing cycle. PCR seamlessly appends the restriction endonuclease recognition domain, and digestion with a class-IIS restriction endonuclease that recognizes the appended recognition domain trims the DNA segment, generating a DNA template composed of a short overhang. Sequencing of a nucleotide in the overhang template occurs through template-directed polymerization with labeled ddNTPs or by template-directed ligation to labeled adaptors. The ligated adaptor sequence that is unique to each cycle allows the PCR step to exponentially regenerate only the desired template precursor during each iteration; thus, this grant tests the hypothesis that exponential DNA amplification in vitro can overcome the background signal accumulation that disables alternative iterative DNA sequencing methods. Also, DNA sequencing occurs in strides up to the number of nucleotides separating the recognition and cleavage domain, maximizing the span of DNA sequenced for a given number of nucleotides. The second specific aim is to develop a protocol to block pre-existing internal class-IIS recognition domains in the DNA segment being sequenced. Using existing instruments and microtiter plates, this method will be able to sequence greater than 1000 vector inserts concurrently, reading greater than 100,000 nucleotides. In the future this method can be adapted to a biochip format. The work proposed in this grant application will allow the sequencing of at least 150 nucleotides in each of thousands of inserts in a DNA library. The sequenced segments will be correct and of sufficient length for extensive bottom up genome sequence reconstruction.