DNA sequence imaging using a Low Energy Electron Microscope

描述（由申请人提供）：对于能够低成本、高质量DNA测序的新技术的开发存在显著需求。基于毛细管阵列电泳和循环阵列测序的既定测序技术提供了这种分析能力，并且下一代商业测序仪以接近10，000美元/基因组的成本提供。一个缺点是，这些技术在一个区段（读段）中仅识别出人类基因组中总共3Gb中的约10至1000个连续碱基对。DNA复杂的重复性质使得完全和准确地重组完整的基因组既昂贵又耗时。 最近，已经提出了透射电子显微镜（TEM）技术，其用重原子（例如，锇），并因此具有显著延长单个读取长度的前景。然而，完整DNA序列的准确测定由于需要标记和关联标记和未标记的碱基而变得复杂。此外，在高分辨率TEM中使用的相对高的电子能量引起辐射损伤，这导致读取错误并限制了可用的电子剂量。 Electron Optica提出开发一种新的电子显微镜，能够以1,000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的双束低能电子显微镜，两束照射的样品具有从0到几个100 eV的能量的电子，和反射的电子被用来形成一个放大的图像。该显微镜包括一个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的图像的潜力。这简化了样品制备并降低了从单个读段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。 拟议的研究将侧重于这一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。将使用最先进的模拟软件对包括像差校正器和单色器在内的列光学器件进行详细分析。将从理论和实验上分析DNA碱基的电子结构，并评估可实现的对比度。拟议的研究将开发一种新的方法，以低成本，高质量的基因组测序需要使基因组信息在个人医疗保健中的使用。 公共卫生相关性：Electron Optica提出开发一种新型的电子显微镜，能够以负担得起的成本阅读无限长度的DNA序列，而不需要用重原子标记碱基，也没有辐射损伤。具有这种能力的仪器使全面的基因组序列信息可用于个人健康护理，并使个性化医疗成为现实。它有望大大提高我们对人类疾病的理解，使诊断成为一个更精确的过程，并为亚纳米分辨率成像在生物科学中的应用开辟了广阔的领域。

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.