Hybridization-Assisted Nanopore DNA Sequencing

描述（由申请人提供）：拟议的研究计划的目的是开发一种低成本的纳米孔测序技术，利用固态纳米孔技术的最新进展，并利用最近提出的杂交辅助纳米孔测序（HANS）的概念。PI及其同事已经提出，通过杂交寡核苷酸的短匹配链（称为DNA探针）并使用纳米孔离子电导来检测探针的位置，可以使用离子电流分布构建DNA序列，从而避免标准杂交测序（SBH）方法中的经典“重复问题”，并且绕过了Kasianowicz等人的原始纳米孔测序方案中对单碱基空间分辨率（0.4nm）的苛刻要求。所提出的HANS测序技术具有快速、低成本和便携的潜力。该研究旨在测试HANS概念的可行性。本项目的具体目标是：（1）使用组合光镊和纳米孔装置确定离子电导方法的空间分辨率。(2)多个纳米孔中DNA易位的平行操作。(3)使用光镊和纳米孔的离子电导检测单链ssDNA上单个DNA探针的杂交。(4)对样品DNA序列进行重新测序以确定准确度。(5)开发用于从头测序的非特异性DNA珠结合技术。 拟议的研究将大大推进生物纳米技术领域，并可能直接导致低成本的DNA测序技术，这反过来又将对美国的公共卫生在疾病检测和潜在疾病的分子过程研究方面产生深远的利益。该研究计划为学生和博士后研究人员提供了极好的培训机会，使他们准备加入新兴生物纳米技术经济的劳动力队伍。

DESCRIPTION (provided by applicant): The objective of the proposed research program is to develop a low-cost nanopore sequencing technology taking advantage of the latest advances in solid-state nanopore technology and utilizing the recently proposed concept of hybridization-assisted nanopore sequencing (HANS). It has been proposed by the PI and coworkers that by hybridizing short matching strands of oligonucleotides, known as DNA probes, and using nanopore ionic conductance to detect the positions of the probes, one can construct DNA sequences using the ionic current profiles, thus avoiding the classical "repeat problem" in the standard sequencing-by-hybridization (SBH) method, and bypassing the harsh requirement of single-base spatial resolution (0.4nm) in the original nanopore sequencing proposal of Kasianowicz et al. The proposed HANS sequencing technology has the potential of being fast, low-cost and portable. The proposed research is to test the feasibility of the HANS concept. The specific aims of the project are: (1) Determine the spatial resolution of the ionic conductance method using a combined optical tweezers and nanopore setup. (2) Parallel manipulation of DNA translocations in multiple nanopores. (3) Detect hybridization of single DNA probes on a single-stranded ssDNA using optical tweezers and ionic conductance of the nanopore. (4) Re-sequencing a sample DNA sequence to determine accuracy. (5) Develop non-specific DNA-bead binding technology for de novo sequencing. The proposed research will greatly advance the field of bio-nanotechnology and could directly result in a low-cost DNA sequencing technology, which in turn will have far-reaching benefits to the public health of the US in disease detection and in the studies of the molecular processes underlying diseases. The research program provides excellent training opportunities for students and postdoctoral researchers in preparing them to join the workforce of the emerging bio-nanotechnology economy.