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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:dcb@seas.harvard.edu
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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

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