Controlling Large DNA Fragments During Nanopore Sequencing

描述（由申请人提供）：纳米孔在DNA测序中的潜在应用已经获得了显著的发展势头。这部分是由于创新的固态技术，但更多的是由于利用生物孔的突破。纳米孔测序的一个前景是非常长的读取长度，然而我们和其他人已经使用短的合成DNA低聚物进行了大多数实验。在这个提议中，我们提出了实验，旨在测试纳米孔如何有效地控制和处理长DNA模板（长达2500 nt），因为它们被DNA聚合酶催化修饰。我们的工作将集中在T7 DNA聚合酶（T7 DNApol）和DNA聚合酶I （KF）的Klenow片段，偶联到α溶血素生物孔（1-HL）。有三个目标：将DNA复制限制在纳米孔中一个接一个捕获的模板链上。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，该策略可以定量地阻断纳米孔中体积相缓冲液中的DNA复制，并激活纳米孔中单个DNA模板的复制。目标2。量化电作用力和DNA/孔相互作用对DNA聚合酶依赖性复制的影响。我们的目标是确定可以在纳米孔上复制的DNA模板的长度。将研究三种条件（见下图），以解决可能影响复制效率的三个独立特性：a)在无负载情况下，纳米孔中捕获的长DNA模板的聚合酶复制；b)依赖聚合酶的长DNA模板复制，以抵抗DNA/孔相互作用产生的阻力；c)依赖聚合酶的复制对抗电阻性电作用力。目标3。以单核苷酸精度确定电压对纳米孔中大DNA模板注册的影响。完整DNA模板的纳米孔测序预先假定在碱基读取期间维持单个核苷酸空间寄存器。对于dna聚合酶控制的易位，这将在每次测量1到100毫秒的范围内。在可能允许聚合酶催化的低电压下，尚不清楚是否可以维持记录。

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.