Sequencing by Recognition

描述（由申请人提供）：氢键介导的电子隧道产生单链DNA碱基组成的高对比度信号，足以使少量重复读取产生的错误率小于万分之一。我们试图将氢键介导的分子识别与DNA易位结合起来，通过纳米孔将每个碱基依次呈现给识别阅读器。这种序列读取器可以以每秒数百到数千个碱基的速度读取基因组DNA，在有限的重复读取次数下具有很高的准确性，并且在一次连续读取中读取序列长度为100,000个碱基（或可能更多）。在这个提议中，我们解决了三个关键问题：(1)我们能否在芯片上复制扫描隧道显微镜（STM）的机械灵活性和精度，从而我们可以复制以前用STM实现的高对比度、高精度化学识别？(2)能否将读取头与纳米孔对齐，读取易位DNA序列？(3)我们能否设计和合成比天然碱基更好的识别分子靶标的“碱基读取器”？我们还将研究其他问题，如二级结构在易位中的作用，以及来自不同读头的数据并置。我们组建了一支具有纳米制造、分子电子学和DNA化学技能的模范团队。我们将：(1)在膜上制造隧道间隙，允许原子尺度的检查，优化间隙制造和化学功能化，以识别核苷单磷酸和DNA二聚体。(2)利用一种新的电沉积工艺将最佳几何形状的间隙与纳米孔对齐。(3)利用磁珠进行操作和位置读出，研究长DNA分子通过这些间隙的易位。(4)设计合成新型“碱基阅读器”。我们将把这些碱基读取试剂提供给社会，以促进探索其他类型的纳米级读出。我们的目标是在五年内展示一个原型仪器，为设计和制造一个可行的商业仪器奠定基础。在本提案中，我们计划开发一种基于“识别测序”的仪器，这种方法的可行性现已得到证明。快速读取长序列DNA的能力对于降低从头测序和重测序的成本至关重要。轻重测序是广泛提供个性化医疗的先决条件，其意义包括更好地规划医疗保健服务、更合适的预防医学和更好地靶向药物。

DESCRIPTION (provided by applicant): Hydrogen-bond mediated electron tunneling yields a high-contrast signal of the base composition of single-stranded DNA, enough so that a small number of repeated reads can yield an error rate smaller than one part in 10,000. We seek to combine hydrogen-bond mediated molecular recognition with DNA translocation through a nanopore to present each base to a recognition reader in turn. Such a sequence reader could read genomic DNA at a speed of hundreds to thousands of bases per second, do so with high accuracy with a limited number of repeated reads and read sequence lengths of 100,000 bases (or possibly more) in one continuous read. In this proposal, we address three key issues: (1) Can we replicate the mechanical flexibility and precision of a scanning tunneling microscope (STM) on a chip, so that we can duplicate the high contrast, high accuracy chemical recognition previously achieved with an STM? (2) Can we align such a reading head with a nanopore so as to read the sequence of translocating DNA? (3) Can we design and synthesize "base-readers" that are better than the native bases at recognizing their molecular targets? We will also examine other issues, such as the role of secondary structure in translocation, and the juxtaposition of data from different reading heads. We have assembled an exemplary team with skills in nanofabrication, molecular electronics and DNA chemistry. We will: (1) Fabricate tunneling gaps on membranes that permit atomic-scale inspection, optimizing the gap fabrication and chemical functionalization for recognition of nucleoside monophosphates and DNA dimers. (2) Use a novel electrodeposition process to align gaps of optimal geometry with nanopores. (3) Study translocation of long DNA molecules through these gaps using magnetic beads both for manipulation and position read-out. (4) Design and synthesize new types of "base-reader". We will make these base-reading reagents available to the community to facilitate exploration of other types of nanoscale readout. We aim to demonstrate a prototype instrument within five years, laying the groundwork for the design and manufacture of a viable commercial instrument. PROJECT HEALTH RELEVANCE In this proposal we plan to develop an instrument based on "Sequencing by Recognition" an approach whose feasibility has now been demonstrated. The ability to read long runs of DNA rapidly is essential to lower the cost of both de novo sequencing and resequencing Facile resequencing is a pre-requisite for the widescale availability of personalized medicine with implications such as better planning of healthcare delivery, more appropriate preventative medicine and better targeting of drugs.