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.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZHG1-HGR-N (M2))
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Schloss, Jeffery
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Electron Optica, Inc.
Palo Alto
United States
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Mankos, M; Shadman, K; Persson, H H J et al. (2014) A novel low energy electron microscope for DNA sequencing and surface analysis. Ultramicroscopy 145:36-49
Mankos, Marian; Shadman, Khashayar (2013) A monochromatic, aberration-corrected, dual-beam low energy electron microscope. Ultramicroscopy 130:13-28