Hostname: page-component-78c5997874-m6dg7 Total loading time: 0 Render date: 2024-11-04T19:31:14.318Z Has data issue: false hasContentIssue false
  • English
  • Français

DNA Base Identification by Electron Microscopy

Published online by Cambridge University Press:  09 October 2012

David C. Bell ,
W. Kelley Thomas ,
Katelyn M. Murtagh ,
Cheryl A. Dionne ,
Adam C. Graham ,
Jobriah E. Anderson  and
William R. Glover
David C. Bell*
Affiliation:
School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA
W. Kelley Thomas
Affiliation:
Hubbard Center for Genome Studies, University of New Hampshire, Durham, NH 03824, USA
Katelyn M. Murtagh
Affiliation:
ZS Genetics, North Reading, MA 01864, USA
Cheryl A. Dionne
Affiliation:
ZS Genetics, North Reading, MA 01864, USA
Adam C. Graham
Affiliation:
Center for Nanoscale Systems, Harvard University, Cambridge, MA 02138, USA
Jobriah E. Anderson
Affiliation:
Hubbard Center for Genome Studies, University of New Hampshire, Durham, NH 03824, USA
William R. Glover
Affiliation:
Hubbard Center for Genome Studies, University of New Hampshire, Durham, NH 03824, USA
*
*Corresponding author:[email protected]
Get access

Abstract

Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.

Keywords

DNAsequencingSTEM
Type
Techniques and Equipment Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 5 , October 2012 , pp. 1049 - 1053
Copyright
Copyright © Microscopy Society of America 2012

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

ASTA. (2010). Advanced Sequencing Technology Awards 2010. September 1, 2010. National Human Genome Research Institute. Available at www.genome.gov/27541189.Google Scholar
Banfalvi, G. & Sarkar, N. (1995). Effect of mercury substitution of DNA on its susceptibility to cleavage by restriction endonucleases. DNA Cell Biol 14, 5.CrossRefGoogle ScholarPubMed
Batson, P.E., Dellby, N. & Krivanek, O.L. (2002). Sub-angstrom resolution using aberration corrected electron optics. Nature 418, 617–620.Google Scholar
Beer, M. & Moudrianakis, E.N. (1962). Determination of base sequence in nucleic acids with the electron microscope: Visibility of a marker. Proc Nat Acad Sci 48, 409–416.CrossRefGoogle ScholarPubMed
Bensimon, A., Simon, A., Chiffaudel, A., Croquette, V., Heslot, F. & Bensimon, D. (1994). Alignment and sensitive detection of DNA by a moving interface. Science 265, 2096–2098.Google Scholar
Bensimon, D., Simon, A.J., Croquette, V. & Bensimon, A. (1995). Stretching DNA with a receding meniscus: Experiments and models. Phys Rev Lett 74, 4754–4757.CrossRefGoogle ScholarPubMed
Bridgman, A.J. & Petersen, G.B. (1996). An improved method for the synthesis of mercurated dUTP. J Sequencing and Mapping 6, 199–209.Google Scholar
Crewe, A.V. (1970). Individual atoms photographed. Science News 97, 524.Google Scholar
Crewe, A.V., Wall, J. & Langmore, J. (1970). Visibility of single atoms. Science 168, 1338–1340.CrossRefGoogle ScholarPubMed
Feynman, R.P. (1999). There is plenty of room at the bottom. In Feynman and Computation, Hey, A.J.G. (Ed.), pp. 63–76. Cambridge, MA: Perseus Press.Google Scholar
Gal-Or, L., Mellema, J.E., Moudrianakis, E.N. & Beer, M. (1967). Electron microscopic study of base sequence in nucleic acids. VII. Cytosine-specific addition of acyl hydrazides. Biochemistry 6(7), 1909–1915.Google Scholar
Jia, C.L., Lentzen, M. & Urban, K. (2003). Atomic resolution imaging of oxygen in perovskite ceramics. Science 299, 870–873.Google Scholar
Livingston, D.C., Dale, R.M.K. & Ward, D.C. (1976). The synthesis and enzymatic polymerization of 5-thio-and 5-methylmercurithio-pyrimidine nucleotides. Biochim Biophys Acta–Nucl Acids Prot Synth 454, 9–20.Google ScholarPubMed
Moudrianakis, E.N. & Beer, M. (1965). Base sequence determination in nucleic acids with the electron microscope, III. Chemistry and microscopy of guanine-labelled DNA. Proc Natl Acad Sci 53, 564–581.Google Scholar
Muller, D.A., Fitting Kourkoutis, L., Murfitt, M., Song, J.H., Hwang, H.Y., Silcox, J., Dellby, N. & Krivanek, O.L. (2008). Atomic-scale chemical imaging of composition and bonding by aberration-corrected microscopy. Science 319, 1073. CrossRefGoogle ScholarPubMed
Ottensmeyer, F.P. (1979). Molecular structure determination by high resolution electron microscopy. Ann Rev Biophys Bioeng 8, 129–144.Google Scholar
Schuster, S.C. (2008). Next-generation sequencing transforms today's biology. Nat Methods 5, 16–18.CrossRefGoogle ScholarPubMed
Villalobos, A., Ness, J.E., Gustafsson, C., Minshull, J. & Govindarajan, S. (2006). Gene designer: A synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics 7, 285.CrossRefGoogle ScholarPubMed
Voyles, P.M., Muller, D.A., Grazul, J.L., Citrin, P.H. & Gossman, H.J.L. (2002). Atomic-scale imaging of individual dopant atoms and clusters in highly n-type bulk Si. Nature 416, 826–829.CrossRefGoogle ScholarPubMed