Controlling Large DNA Fragments During Nanopore Sequencing

描述(由申请人提供)：纳米孔用于DNA测序的潜在用途已经获得了显著的势头。这在一定程度上是由于创新的固态技术，但更多的是由于利用生物毛孔的突破。纳米孔测序的一个希望是非常长的读取长度，然而我们和其他人使用短的合成DNA寡聚体进行了大多数实验。在这个建议中，我们提供了一些实验，旨在测试当DNA聚合酶催化修饰长DNA模板时，纳米孔控制和处理长DNA模板(最长可达2500个核苷酸)的效率。我们的工作将集中在T7DNA聚合酶(T7DNApol)和DNA聚合酶I(KF)的Klenow片段上，与α溶血素生物孔(1-HL)偶联。有三个目标：目标1.将DNA复制限制在模板链上，这些模板链在纳米孔中逐个捕获。为了确保在催化过程中对单个DNA模板进行有效的序列分析，我们将优化我们实验室开发的一种新策略，该策略定量地阻止沐浴纳米孔的块状相缓冲区中的DNA复制，并仅在纳米孔激活单个DNA模板的复制。目的2.量化电场力和DNA/孔相互作用对DNA聚合酶依赖的复制的影响。我们的目标是确定可在纳米孔上重复复制的DNA模板的长度。将考察三个条件(见下图)以解决可能影响复制效率的三个独立性质：a)在无负载的情况下捕获在纳米孔中的长DNA模板的聚合酶复制；b)依赖聚合酶的长DNA模板的复制，以对抗DNA/孔相互作用产生的阻力；c)针对阻性电势的聚合酶依赖的复制。目的3.以单核苷酸精确度确定电压对纳米孔中大DNA模板注册量的影响。完整DNA模板的纳米孔测序前提是在读取碱基的时间内保持单核苷酸空间注册。对于DNA聚合酶控制的易位，这将在每次测量的1到100毫秒的范围内。在可能允许聚合酶催化的低电压下，是否能保持注册尚不清楚。 与公共卫生相关：高速DNA测序是了解包括癌症和心脏病在内的人类疾病的基础。这项建议解决了一种基于生物纳米孔的有前景的新DNA测序技术的基本问题。

DESCRIPTION (provided by applicant): The potential use of nanopores for DNA sequencing has gained significant momentum. This is partly due to innovative solid state techniques, but more so due to breakthroughs using biological pores. One promise of nanopore sequencing has been very long read lengths, however we and others have performed most of our experiments using short synthetic DNA oligomers. In this proposal, we present experiments designed to test how efficiently nanopores can control and process long DNA templates (up to 2500 nt in length) as they are catalytically modified by DNA polymerases. Our work will focus on T7 DNA polymerase (T7 DNApol) and the Klenow fragment of DNA polymerase I (KF), coupled to the alpha hemolysin biopore (1-HL). There are three aims: Aim 1. Limit DNA replication to template strands captured one-by-one in the nanopore. To ensure efficient serial analysis of individual DNA templates during catalysis, we will optimize a new strategy developed in our laboratory that quantitatively blocks DNA replication in bulk phase buffer bathing the nanopore, and that activates replication of individual DNA templates exclusively at the nanopore. Aim 2. Quantify the effect of electrical force and DNA/pore interactions on DNA polymerase- dependent replication. Our objective is to determine the length of DNA template that can be reproducibly replicated on the nanopore. Three conditions (see figure below) will be examined to address three independent properties that could influence replication efficiency: a) Polymerase replication of long DNA templates captured in the nanopore under no load; b) Polymerase dependent replication of long DNA templates against resistive forces that arise from DNA/pore interactions; c) Polymerase dependent replication against a resistive electrical force. Aim 3. Determine the effect of voltage on registry of large DNA templates in the nanopore at single nucleotide precision. Nanopore sequencing of intact DNA templates presupposes maintenance of single nucleotide spatial register during the time a base is read. For DNA-polymerase-controlled translocation this would be in the range of 1 to 100 milliseconds per measurement. At low voltages that are likely to permit polymerase catalysis, it is unclear if registry can be maintained. PUBLIC HEALTH RELEVANCE: High speed DNA sequencing is fundamental to understanding human diseases including cancer and heart disease. This proposal addresses fundamental questions about one promising new DNA sequencing technique based on biological nanopores.