Exploration of Solid-state Nanopore Reading Labeled Linear DNA Sequence

描述（由申请人提供）：在本提案中，我们研究了纳米孔测序概念的关键组成部分。虽然纳米孔测序概念的最终目标是在不复制DNA和不结合标记的情况下读取核苷酸的线性序列，但在这里，我们探索了通过结合生物素标记的核苷酸来读取DNA的线性序列。我们计划结合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。

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.