REACTIVE SEQUENCING OF DNA

描述：(研究人员摘要)DNA测序的一种新方法是 提出的，潜在的应用于大规模基因组测序 以及小规模的诊断性测序。带退火引物的DNA模板 将被拴在琼脂糖珠表面并在 存在无核酸外切酶的DNA聚合酶，每个DNA聚合酶都有单独的流动 四种脱氧核苷酸三磷酸(dNTP‘s)，荧光素标记在 非碱基配对位点。因为聚合酶对 DNTP，与引物延伸处的模板碱基互补 酶切位点的聚合酶反应只会发生在 提供了正确的dNTP；通过检测到已发生扩展并知道 所提供的dNTP的身份，则可以读取该序列。事实是，只有 一种类型的dNTP在任何时候都与引物/模板双链接触 一次确保底物链的延伸同步进行 来自所有链的延伸信号反映了碱基同一性 完全相同的模板站点。扩展将通过检测 过量未反应后被掺入的脱氧核苷酸的荧光 DNTP已经被冲走了。为了能够继续检测到进一步的 延长，结合的荧光素标记将被光化学处理 在每次读数后销毁。几百个碱基的序列读出长度 都是意料之中的。聚合酶保真度和光化学损伤的问题 可能会影响序列准确性和读取长度，将进行调查， 化学和测序程序将进行调整，以优化，首先， 序列读取长度，以及随后的测序速度。初赛 将建造能够半自动测序的毛细管流动装置。 这种新的测序方法可能会与传统的测序方法竞争 用于基因组测序应用的方法，尤其是针对 允许对目标短区域进行快速且廉价的诊断测序 人类基因组编码与人类疾病有关的蛋白质。

DESCRIPTION: (Investigator's Abstract)A new approach to DNA sequencing is proposed, with potential applications both to large-scale genomic sequencing and to small-scale diagnostic sequencing. DNA templates with annealed primers will be tethered to agarose bead surfaces and interrogated serially, in the presence of exonuclease-free DNA polymerase, with separate flows of each of the four deoxynucleotide triphosphates (dNTP's), labeled with fluorescein at non-base-pairing sites. Because the polymerase enzyme is highly selective for the dNTP which is complementary to the template base at the primer extension site, the polymerase reaction to extend the primer will only occur when the correct dNTP is supplied; by sensing that extension has occurred and knowing the identity of the dNTP supplied, the sequence may be read. The fact that only a single type of dNTP is in contact with the primer/template duplexes at any one time ensures that extension of the primer strands proceeds synchronously so that the extension signals from all the strands reflect the base identity at exactly the same template sites. Extension will be sensed by detecting the fluorescence from the incorporated deoxynucleotides after excess unreacted dNTP's have been rinsed away. In order to allow continued detection of further extensions, the incorporated fluorescein labels will be photochemically destroyed after each readout. Sequence read lengths of several hundred bases are anticipated. Issues of polymerase fidelity and photochemical damage that can affect sequence accuracy and read length will be investigated, and the chemistry and the sequencing procedures will be adjusted to optimize, first, sequence read length, and subsequently, sequencing speed. A preliminary capillary flow device capable of semi-automated sequencing will be constructed. This novel new sequencing method may compete with conventional sequencing methods for genomic sequencing applications and in particular is aimed at allowing rapid and inexpensive diagnostic sequencing of targeted short regions of the human genome which code for proteins implicated in human disease.