REACTIVE SEQUENCING OF DNA

描述：（研究者摘要）一种新的DNA测序方法是 提出，与潜在的应用，大规模基因组测序 以及小规模诊断测序。退火引物DNA模板 将被拴系到琼脂糖珠表面并连续询问， 无核酸外切酶的DNA聚合酶的存在，每种聚合酶都有单独的流动， 四个脱氧核苷酸三磷酸（dNTP），用荧光素标记， 非碱基配对位点。因为聚合酶对 在引物延伸处与模板碱基互补的dNTP 位点时，延伸引物的聚合酶反应将仅在 提供正确的dNTP;通过感测已经发生的延伸并知道 所提供的dNTP的身份，可以读取序列。事实上只有 单一类型的dNTP与引物/模板双链体在任何位置接触， 一次确保引物链的延伸同步进行 来自所有链的延伸信号反映了碱基的同一性， 完全相同的模板网站。将通过检测 在过量未反应的脱氧核苷酸后， dNTP已经被冲洗掉了为了能够继续探测到 延伸，掺入的荧光素标记将被光化学地 在每次读取后销毁。数百个碱基的序列读取长度 都在预料之中。聚合酶保真度和光化学损伤的问题， 会影响序列准确度和读长， 化学和测序程序将被调整以优化，首先， 序列读取长度，以及随后的测序速度。初步 将构建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，特别是针对 允许对靶向短区域进行快速和廉价的诊断测序 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.