REACTIVE SEQUENCING OF DNA

DNA 反应测序

• 批准号: 6526724
• 负责人: PETER WILLIAMS
• 金额: $ 15.25万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-07 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6526724
• 关键词: DNA; DNA directed DNA polymerase; DNA primers; biomedical automation; biotechnology; deoxyribonucleoside triphosphate; diagnosis quality /standard; exonuclease; fluoresceins; genome; high throughput technology; mass spectrometry; microarray technology; nucleic acid sequence; polymerase chain reaction; sequence tagged sites; technology /technique development

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.

描述：（研究者摘要）一种新的 DNA 测序方法是 提出，具有大规模基因组测序的潜在应用 以及小规模诊断测序。带有退火引物的 DNA 模板 将被拴在琼脂糖珠表面并连续询问， 存在无核酸外切酶的 DNA 聚合酶，每个聚合酶具有单独的流动 四种脱氧核苷酸三磷酸（dNTP），用荧光素标记 非碱基配对位点。因为聚合酶具有高度选择性 dNTP 与引物延伸处的模板碱基互补 位点，延伸引物的聚合酶反应仅在以下情况下发生： 提供了正确的 dNTP；通过感知延伸已经发生并知道 提供的 dNTP 的身份，可以读取序列。事实是只有 单一类型的 dNTP 在任意位置与引物/模板双链体接触 一次确保引物链的延伸同步进行，以便 来自所有链的延伸信号反映了碱基身份 完全相同的模板网站。扩展将通过检测来感测 过量未反应后掺入的脱氧核苷酸发出荧光 dNTP 已被冲洗掉。为了能够继续检测进一步 扩展时，合并的荧光素标记将发生光化学反应 每次读出后销毁。数百个碱基的序列读取长度 是预期的。聚合酶保真度和光化学损伤的问题 会影响序列准确性和阅读长度将被调查，并且 将调整化学和测序程序以优化，首先， 序列读取长度，以及随后的测序速度。初步 将建造能够半自动测序的毛细管流动装置。 这种新颖的测序方法可能会与传统测序竞争 用于基因组测序应用的方法，特别是针对 允许对目标短区域进行快速且廉价的诊断测序 人类基因组的一部分，编码与人类疾病有关的蛋白质。