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DNA POLYMERASES FOR SINGLE MOLECULE DNA SEQUENCING

用于单分子 DNA 测序的 DNA 聚合酶

基本信息

批准号：
6294220
负责人：
JOHN G. K. WILLIAMS
金额：
$ 10万
依托单位：
LI-COR BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-01-01 至 2001-06-30
项目状态：
已结题

项目摘要

APPLICANT'S DESCRIPTION: DNA polymerases with altered substrate specificity are needed to support a new approach to single molecule DNA sequencing. Single molecule methods can be applied to DNA isolated directly from a subject organism, eliminating the need to clone, map and sort DNA fragments prior to sequencing. The need for electrophoretic separation is eliminated and reagent consumption is minimal. Single molecule detection has been developed and practiced by physicists and chemists for over 10 years. The crucial optical and detection systems needed for single molecule sequencing now exist. To implement this technology, novel nucleotides labeled at two different atomic positions have been synthesized and tested as substrates for DNA polymerases. Whereas some enzymes utilize nucletides labeled at the first position and other enzymes utilize nucleotides labeled at the second position, we could not identify any natural polymerase able to efficiently incorporate nucleotides labeled at both positions (double-labeled nucleotides). In Phase I, we will develop methods for the directed evolution of DNA polymerases adapted to the utilization of double-labeled nucleotides. Directed evolution will be used in Phase II to select for highly processive DNA polymerases having the desired substrate specificities. PROPOSED COMMERCIAL APPLICATION: A genomic DNA sequencing technology that reduces cost and eliminates the need for cloning, physical mapping and electrophoresis would have great appeal to both the academic and industrial genomic research communities.

申请人的描述：具有改变的底物特异性的DNA聚合酶是需要支持一种新的单分子 DNA 测序方法。单身的分子方法可应用于直接从受试者中分离的 DNA 生物体，无需在之前克隆、绘制和分类 DNA 片段测序。无需电泳分离，并且无需试剂消耗是最小的。单分子检测已被开发并物理学家和化学家已经实践了 10 多年。关键的光学和单分子测序所需的检测系统现已存在。实施这项技术在两个不同的原子位置标记了新的核苷酸已作为 DNA 聚合酶的底物进行合成和测试。然而一些酶利用在第一个位置标记的核苷酸，而其他酶则利用利用第二个位置标记的核苷酸，我们无法识别任何天然聚合酶能够有效地掺入标记的核苷酸位置（双标记核苷酸）。在第一阶段，我们将开发方法 DNA聚合酶的定向进化适应了利用双标记核苷酸。定向进化将在第二阶段使用选择具有所需底物的高持续性 DNA 聚合酶特殊性。拟议的商业应用：一种基因组 DNA 测序技术，可降低成本并消除对克隆、物理作图和电泳对人类和人类都具有巨大的吸引力。学术和工业基因组研究团体。