Exploration of Solid-state Nanopore Reading Labeled Linear DNA Sequence

描述（由申请人提供）： 在这个提议中，我们研究了纳米孔测序概念的一个关键组成部分。虽然纳米孔测序概念的最终目标是在不复制DNA和不掺入标记的情况下读取核苷酸的线性序列，但是在这里，我们探索通过掺入生物素标记的核苷酸来阅读DNA的线性序列。我们计划加入生物素标记的核苷酸，以更好地区分所有四种核苷酸之间的信号差异，并进行读取DNA线性序列所需的基础研究和技术开发。 该策略是首先通过在其他天然核苷酸存在下将生物素标记的核苷酸（BLN）掺入四个碱基之一中来增加DNA中碱基之间的信号差异。例如，生物素标记的鸟嘌呤（BLG）将被掺入DNA中，然后我们将通过单碱基分辨率纳米孔传感系统检测BLG的位置。我们将研究检测BLN在单链或双链DNA分子中的位置的能力。具有全部相同类型的被BLN（例如全部G（xj））取代的核苷酸的一个DNA分子将以受控的速度移动通过电解质溶液中的电压偏置的固态纳米孔。易位的DNA将部分地阻断纳米孔中的离子电流流动，并且产生的电流阻断信号将区分生物素标记的G（xj）和其余碱基之间的差异作为电流信号GI（tj），这里t是时间。因此，通过校准xj和tj之间的关系，G（x1），G（x2），.，可以从GI（tj）估计G（xj）。其他三种类型的核苷酸A（xj）、T（xj）和C（xj）的位置可以以相同的方式确定。因此，可以确定DNA分子的整个序列。我们最初的目标是测序一个约1000个碱基对（bp）的DNA分子，其序列是已知的。 这将使我们能够开发基于固态纳米孔的测序所需的基本技术。 该提案的具体目标是： 1.制造和测试单基分辨率（厚度为1- 2nm）固态纳米孔。 2.开发控制DNA通过纳米孔移位的速率的方法， ~100 μ s/base。 3.优化我们的纳米孔传感系统和数据分析程序，以确定 BLN（xj）在~1000个碱基长的DNA中。研究高分辨率固态 纳米孔装置可以确定BLN（xj）在DNA中的位置。 在这个提案中，我们调查了纳米孔测序概念的一个关键组成部分。虽然纳米孔测序概念的最终目标是在不复制DNA和不掺入标记的情况下读取核苷酸的线性序列，但是在这里，我们探索通过掺入生物素标记的核苷酸来阅读DNA的线性序列。我们计划加入生物素标记的核苷酸，以更好地区分所有四种核苷酸之间的信号差异，并进行读取DNA线性序列所需的基础研究和技术开发。 该策略是首先通过在其他天然核苷酸存在下将生物素标记的核苷酸（BLN）并入四个碱基之一中来增加DNA中碱基之间的信号差异，然后我们将通过单碱基分辨率纳米孔传感系统检测BLG的位置。我们将研究检测BLN在单链或双链DNA分子中的位置的能力。我们最初的目标是测序一个约1000个碱基对（bp）的DNA分子，其序列是已知的。这将使我们能够开发基于固态纳米孔的测序所需的基本技术。

DESCRIPTION (provided by applicant): In this proposal, we investigate a key component of the nanopore sequencing concept. Although the ultimate goal of the nanopore sequencing concept is to read the linear sequence of nucleotides without copying the DNA and without incorporating of labels, here, we explore of reading the linear sequence of a DNA by incorporating biotin labeled nucleotides. We plan to incorporate biotin labeled nucleotides to better differentiate the signal difference between all four nucleotides and conduct the basic research and technology development that are needed to read the linear sequence of a DNA. The strategy is to first increase the signal difference between bases in a DNA by incorporating Biotin-Labeled Nucleotides (BLN) into one of the four bases in the presence of the other natural nucleotides. For example, Biotin-Labeled Guanine (BLG) will be incorporated into DNA and then we will detect the locations of BLG by a single base resolution nanopore sensing system. We will investigate the ability to detect the positions of BLN in a single-stranded or double-stranded DNA molecule. One DNA molecule with all the same type of nucleotides substituted by a BLN, all G(xj) for example, will move at a controlled speed through a voltage biased solid state nanopore in an electrolyte solution. The translocating DNA will partially block the ionic current flow in the nanopore, and the current blockage signal generated will distinguish the difference between the biotin-labeled G(xj) and the rest of the bases as an electrical current signal GI(tj), here t is the time. Thus by calibrating the relation between xj and tj, the locations of G(x1), G(x2), ..., G(xj) can be estimated from GI(tj). The locations of the other three types nucleotides A(xj), T(xj), and C(xj) can be determined the same way. Thus, the whole sequence of a DNA molecule could be determined. Our initial goal is to sequence a DNA molecule of ~1000 base pair (bp) whose sequence is known. This will permit us to develop the basic technology necessary for solid-state nanopore-based sequencing. The specific goals in this proposal are: 1. Fabricate and test single base resolution (1-2 nm in thickness) solid-state nanopores. 2. Develop methods to control the rate of DNA translocation through a nanopore at ~100¿s/base. 3. Optimize our nanopore sensing system and data analysis routines to determine the BLN(xj) in a ~1000 base long DNA. Study how accurate a high resolution solid-state nanopore device can determine the location of BLN(xj) in DNA. PROJECT HEALTH RELEVANCE In this proposal, we investigate a key component of the nanopore sequencing concept. Although the ultimate goal of the nanopore sequencing concept is to read the linear sequence of nucleotides without copying the DNA and without incorporating of labels, here, we explore of reading the linear sequence of a DNA by incorporating biotin labeled nucleotides. We plan to incorporate biotin labeled nucleotides to better differentiate the signal difference between all four nucleotides and conduct the basic research and technology development that are needed to read the linear sequence of a DNA. The strategy is to first increase the signal difference between bases in a DNA by incorporating Biotin-Labeled Nucleotides (BLN) into one of the four bases in the presence of the other natural nucleotides and then we will detect the locations of BLG by a single base resolution nanopore sensing system. We will investigate the ability to detect the positions of BLN in a singlestranded or double-stranded DNA molecule. Our initial goal is to sequence a DNA molecule of ~1000 base pair (bp) whose sequence is known. This will permit us to develop the basic technology necessary for solid-state nanopore-based sequencing.