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DNA Base Identification by Electron Microscopy

  • David C. Bell (a1), W. Kelley Thomas (a2), Katelyn M. Murtagh (a3), Cheryl A. Dionne (a3), Adam C. Graham (a4), Jobriah E. Anderson (a2) and William R. Glover (a2)...
Abstract
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.

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*Corresponding author:dcb@seas.harvard.edu
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G. Banfalvi & N. Sarkar (1995). Effect of mercury substitution of DNA on its susceptibility to cleavage by restriction endonucleases. DNA Cell Biol 14, 5.

P.E. Batson , N. Dellby & O.L. Krivanek (2002). Sub-angstrom resolution using aberration corrected electron optics. Nature 418, 617620.

M. Beer & E.N. Moudrianakis (1962). Determination of base sequence in nucleic acids with the electron microscope: Visibility of a marker. Proc Nat Acad Sci 48, 409416.

A. Bensimon , A. Simon , A. Chiffaudel , V. Croquette , F. Heslot & D. Bensimon (1994). Alignment and sensitive detection of DNA by a moving interface. Science 265, 20962098.

D. Bensimon , A.J. Simon , V. Croquette & A. Bensimon (1995). Stretching DNA with a receding meniscus: Experiments and models. Phys Rev Lett 74, 47544757.

A.J. Bridgman & G.B. Petersen (1996). An improved method for the synthesis of mercurated dUTP. J Sequencing and Mapping 6, 199209.

A.V. Crewe , J. Wall & J. Langmore (1970). Visibility of single atoms. Science 168, 13381340.

L. Gal-Or , J.E. Mellema , E.N. Moudrianakis & M. Beer (1967). Electron microscopic study of base sequence in nucleic acids. VII. Cytosine-specific addition of acyl hydrazides. Biochemistry 6(7), 19091915.

C.L. Jia , M. Lentzen & K. Urban (2003). Atomic resolution imaging of oxygen in perovskite ceramics. Science 299, 870873.

D.C. Livingston , R.M.K. Dale & D.C. Ward (1976). The synthesis and enzymatic polymerization of 5-thio-and 5-methylmercurithio-pyrimidine nucleotides. Biochim Biophys Acta–Nucl Acids Prot Synth 454, 920.

E.N. Moudrianakis & M. Beer (1965). Base sequence determination in nucleic acids with the electron microscope, III. Chemistry and microscopy of guanine-labelled DNA. Proc Natl Acad Sci 53, 564581.

D.A. Muller , L. Fitting Kourkoutis , M. Murfitt , J.H. Song , H.Y. Hwang , J. Silcox , N. Dellby & O.L. Krivanek (2008). Atomic-scale chemical imaging of composition and bonding by aberration-corrected microscopy. Science 319, 1073.

F.P. Ottensmeyer (1979). Molecular structure determination by high resolution electron microscopy. Ann Rev Biophys Bioeng 8, 129144.

S.C. Schuster (2008). Next-generation sequencing transforms today's biology. Nat Methods 5, 1618.

A. Villalobos , J.E. Ness , C. Gustafsson , J. Minshull & S. Govindarajan (2006). Gene designer: A synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics 7, 285.

P.M. Voyles , D.A. Muller , J.L. Grazul , P.H. Citrin & H.J.L. Gossman (2002). Atomic-scale imaging of individual dopant atoms and clusters in highly n-type bulk Si. Nature 416, 826829.

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Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
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