First Nucleotide Sequence Data from an Electron Microscopy Based DNA Sequencer

Own, Christopher S.; Bleloch, Andrew; Lerach, W.; Bowell, Charlotte; Hamalainen, Mark; Herschleb, Jill; Melville, Claire M.; Stark, Jeremy M.; Andregg, Michael; Andregg, William

doi:10.1017/s1431927613003036

Cited by 5 publications

(6 citation statements)

References 6 publications

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“…Transmission Electron Microscopy (TEM) has been explored for imaging long DNA segments [ 1 , 2 ] by utilizing high electron energies (80–300 keV) to achieve sub-nanometer resolution. The high impact energy, however, not only produces radiation damage, it necessitates the use of heavy atom labels to provide contrast in the image of the nucleotides.…”

Section: Introductionmentioning

confidence: 99%

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy

et al. 2016

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DNA sequencing by imaging in an electron microscope is an approach that holds promise to deliver long reads with low error rates and without the need for amplification. Earlier work using transmission electron microscopes, which use high electron energies on the order of 100 keV, has shown that low contrast and radiation damage necessitates the use of heavy atom labeling of individual nucleotides, which increases the read error rates. Other prior work using scattering electrons with much lower energy has shown to suppress beam damage on DNA. Here we explore possibilities to increase contrast by employing two methods, X-ray photoelectron and Auger electron spectroscopy. Using bulk DNA samples with monomers of each base, both methods are shown to provide contrast mechanisms that can distinguish individual nucleotides without labels. Both spectroscopic techniques can be readily implemented in a low energy electron microscope, which may enable label-free DNA sequencing by direct imaging.

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Section: Introductionmentioning

confidence: 99%

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy

et al. 2016

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“…For applications where single-strandedness is crucial, as in EM-based DNA sequencing, the strands must be very long and extraction lengths scaled commensurately to ensure that the desired quantity of single-strand segments are deposited. At higher EM resolution, quantification of single-strandedness, base-to-base stretching, and observation of individual nucleotide labeling is possible, though this is beyond the scope of this paper and is discussed elsewhere [24], [25]. However, several qualitative features of threading that can be discerned from high-resolution images are examined in Figure S6 .…”

Section: Discussionmentioning

confidence: 99%

Molecular Threading: Mechanical Extraction, Stretching and Placement of DNA Molecules from a Liquid-Air Interface

Payne

Andregg²,

Kemmish³

et al. 2013

PLoS ONE

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We present “molecular threading”, a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA deposited in arrays onto elastomeric surfaces and thin membranes. Molecular threading demonstrates high straightness and uniformity over length scales from nanometers to micrometers, and represents an alternative to existing DNA deposition and linearization methods. These results point towards scalable and high-throughput precision manipulation of single-molecule polymers.

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Section: Dna Metallization Via Metal Np Staining and Nanowire Growthmentioning

confidence: 99%

“…[ 160 ] While individual bases have been modified in the above studies as a proof‐of‐concept, multiplexing with different contrast‐agent labels in combination with a high‐resolution automated imager allows for direct DNA sequencing from EM‐based images, as demonstrated by Own et al, shown in Figure 12C. [ 161 ]…”

Section: Dna Metallization Via Metal Np Staining and Nanowire Growthmentioning

confidence: 99%

Toward Visualizing Genomic DNA Using Electron Microscopy via DNA Metallization

et al. 2023

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Figure 5. DNA-templated silver nanowire. The top left image is the electrode pattern used for silver nanowire assembly. A) Thiolated oligonucleotides complementary to the sticky ends of λ DNA are attached to the electrodes. B) λ DNA bridge connects the two gold electrodes. C) Silver ions are loaded on the DNA bridge. D) A silver nanowire is assembled by chemical reduction. E) Metallic silver aggregates on the λ DNA. Reproduced with permission. [55]

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First Nucleotide Sequence Data from an Electron Microscopy Based DNA Sequencer

Abstract: Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Cited by 5 publications

References 6 publications

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy

Molecular Threading: Mechanical Extraction, Stretching and Placement of DNA Molecules from a Liquid-Air Interface

Toward Visualizing Genomic DNA Using Electron Microscopy via DNA Metallization

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