DNA sequence imaging using a Low Energy Electron Microscope

使用低能电子显微镜进行 DNA 序列成像

• 批准号: 8183159
• 负责人: Marian Mankos
• 金额: $ 24.96万
• 依托单位: ELECTRON OPTICA, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8183159
• 关键词: Base Pairing; Base Sequence; Blood capillaries; Collaborations; Complex; Cost aspects; DNA; DNA Sequence; Data Analyses; Development; Diagnosis; Dose; Electron Microscope; Electron Microscopy; Electrons; Electrophoresis; Ensure; Genome; Genomics; Goals; Healthcare; Hour; Human Genome; Image; Image Analysis; Imaging Techniques; Imaging technology; Individual; Label; Length; Measures; Medicine; Microscope; Modeling; Nature; Nucleotides; Optics; Osmium; Performance; Phase; Preparation; Procedures; Property; Radiation; Reading; Research; Resolution; Sampling; Simulate; Speed; System; Techniques; Technology; Time; Transmission Electron Microscopy; base; capillary; cost; design; electron optics; electronic structure; genome sequencing; human disease; improved; instrument; meetings; nanometer; nanoscale; new technology; next generation; novel; novel strategies; prototype; simulation software; tool

DESCRIPTION (provided by applicant): Significant demand exists for the development of novel technologies capable of low-cost, high quality DNA sequencing. Established sequencing techniques based on capillary array electrophoresis and cyclic array sequencing offer such analytical capability, and next generation commercial sequencers deliver at a cost approaching $10,000/genome. One drawback is that these technologies identify in one segment (read) only about 10 to 1000 sequential base pairs out of the total 3 Gb in the human genome. The complex repetitive nature of DNA makes it costly and time consuming to completely and accurately reassemble a full genome. Recently, transmission electron microscopy (TEM) techniques have been proposed that label specific DNA bases with heavy atoms (e.g., osmium) and thus have the promise of significantly extending the length of individual reads. However, the accurate determination of the complete DNA sequence is complicated by the need for labeling and correlating the labeled and unlabeled bases. In addition, the relatively high electron energy used in high resolution TEMs causes radiation damage that leads to read errors and limits the usable electron dose. Electron Optica proposes to develop a novel electron microscope capable of imaging a DNA base sequence of unlimited length at a cost of $1,000/genome with the high accuracy needed for full-scale sequencing. In this technique, which we call monochromatic aberration-corrected dual-beam low energy electron microscopy, two beams illuminate the sample with electrons having energies from 0 to a few 100 eV, and the reflected electrons are utilized to form a magnified image. The microscope includes a monochromator and aberration corrector, and has the potential of delivering images of unlabeled DNA with nucleotide-specific contrast. This simplifies sample preparation and eases the computational complexity needed to assemble the sequence from individual reads. In addition, at low landing energies there is no radiation damage, so high electron doses needed for high throughput and low cost can be used. The proposed research will focus on the feasibility of the key aspects required for this approach, i.e., achieving high spatial resolution, high throughput and DNA base-specific contrast. A detailed analysis of the column optics including the aberration corrector and monochromator will be performed using state-of-the-art simulation software. Analysis of the electronic structure of DNA bases will be carried out theoretically and experimentally and the achievable contrast will be evaluated. The proposed research will develop a new approach to low cost, high quality genome sequencing needed to enable the use of genomic information in individual health care. PUBLIC HEALTH RELEVANCE: Electron Optica proposes to develop a novel electron microscope capable of reading DNA sequences of unlimited length at affordable cost without labeling the bases with heavy atoms and without radiation damage. An instrument of such capability makes comprehensive genomic sequence information available for individual health care and makes personalized medicine a reality. It promises to greatly improve our understanding of human diseases, make diagnosis a more precise procedure and opens a wide field of applications for sub-nanometer resolution imaging in the biosciences.

描述（由申请人提供）：对低成本、高质量DNA测序的新技术的开发存在巨大需求。基于毛细管阵列电泳和循环阵列测序的现有测序技术提供了这种分析能力，下一代商用测序仪的成本接近10,000美元/基因组。一个缺点是，这些技术在一个片段（读取）中只能从人类基因组的总3gb中识别出大约10到1000个序列碱基对。DNA复杂的重复性使得完全准确地重组一个完整的基因组既昂贵又耗时。最近，透射电子显微镜（TEM）技术被提出用重原子（如锇）标记特定的DNA碱基，从而有希望显著延长单个读取的长度。然而，准确测定完整的DNA序列是复杂的，因为需要标记和关联标记和未标记的碱基。此外，在高分辨率tem中使用的相对较高的电子能量会导致辐射损伤，从而导致读取错误并限制可用电子剂量。Electron Optica提议开发一种新型电子显微镜，能够以1000美元/基因组的成本对无限长度的DNA碱基序列进行成像，并具有全面测序所需的高精度。在这种技术中，我们称之为单色像差校正双光束低能量电子显微镜，两束用能量从0到几个100 eV的电子照射样品，反射的电子被用来形成放大的图像。该显微镜包括单色仪和像差校正器，并具有提供具有核苷酸特异性对比度的未标记DNA图像的潜力。这简化了样品制备，并减轻了从单个读取组装序列所需的计算复杂性。此外，在低着陆能量下没有辐射损伤，因此可以使用高通量和低成本所需的高电子剂量。本研究将重点关注该方法所需的关键方面的可行性，即实现高空间分辨率、高通量和DNA碱基特异性对比度。柱光学包括像差校正器和单色器的详细分析将使用最先进的模拟软件进行。对DNA碱基的电子结构进行理论和实验分析，并对可实现的对比进行评估。拟议的研究将开发一种低成本、高质量的基因组测序的新方法，以实现在个人保健中使用基因组信息。