Previous Article |
Table of Contents
| Next Article 
Vol. 95, Issue 14, 8046-8051, July 7, 1998
* W. M. Keck Laboratory for Biomolecular Imaging, Department of
Chemistry, New York University, 31 Washington Place, New York, NY
10003; and Communicated by S. Walter Englander, University of Pennsylvania
School of Medicine, Swarthmore, PA, April 23, 1998 (received for review February 15, 1998)
New mapping approaches construct ordered restriction maps from
fluorescence microscope images of individual, endonuclease-digested DNA
molecules. In optical mapping, molecules are elongated and fixed onto
derivatized glass surfaces, preserving biochemical accessibility and
fragment order after enzymatic digestion. Measurements of relative
fluorescence intensity and apparent length determine the sizes of
restriction fragments, enabling ordered map construction without
electrophoretic analysis. The optical mapping system reported here is
based on our physical characterization of an effect using fluid flows
developed within tiny, evaporating droplets to elongate and fix DNA
molecules onto derivatized surfaces. Such evaporation-driven molecular
fixation produces well elongated molecules accessible to restriction
endonucleases, and notably, DNA polymerase I. We then developed the
robotic means to grid DNA spots in well defined arrays that are
digested and analyzed in parallel. To effectively harness this effect
for high-throughput genome mapping, we developed: (i)
machine vision and automatic image acquisition techniques to work with
fixed, digested molecules within gridded samples, and
(ii) Bayesian inference approaches that are used to
analyze machine vision data, automatically producing high-resolution
restriction maps from images of individual DNA molecules. The aggregate
significance of this work is the development of an integrated system
for mapping small insert clones allowing biochemical data obtained from
engineered ensembles of individual molecules to be automatically
accumulated and analyzed for map construction. These approaches are
sufficiently general for varied biochemical analyses of individual
molecules using statistically meaningful population sizes.
Copyright © 1998 by The National Academy of Sciences 0027-8424/98/958046-6$2.00/0
Biophysics
Automated high resolution optical mapping using arrayed,
fluid-fixed DNA molecules
,,
,
,,,**
Courant Institute of Mathematical Sciences,
Department of Computer Science, New York University, 251 Mercer Street,
New York, NY 10003
J.J. and J.R. contributed equally to this work.
Present address: Johns Hopkins School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205.
§
Present address: CuraGen Corporation, 322 East Main Street,
Branford, CT 06405.
¶
Present address: VirtuFlex Software Corporation, 930 Massachusetts Avenue, Cambridge, MA 02139.
**
To whom reprint requests should be addressed at: W. M. Keck
Laboratory for Biomolecular Imaging, Department of Chemistry, New York
University, Room 866, 31 Washington Place, New York, NY 10003. e-mail:
schwad01{at}mcrcr.med.nyu.edu.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg What's this?
This article has been cited by other articles in HighWire Press-hosted journals:
|
|
 |
|
  K. Jo, D. M. Dhingra, T. Odijk, J. J. de Pablo, M. D. Graham, R. Runnheim, D. Forrest, and D. C. Schwartz A single-molecule barcoding system using nanoslits for DNA analysis PNAS, February 20, 2007; 104(8): 2673 - 2678. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Grossman Paths toward Algal Genomics Plant Physiology, February 1, 2005; 137(2): 410 - 427. [Full Text] [PDF]
|
|
|
|
|
|
X. Qu, D. Wu, L. Mets, and N. F. Scherer Nanometer-localized multiple single-molecule fluorescence microscopy PNAS, August 3, 2004; 101(31): 11298 - 11303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Gordon, T. Ha, and P. R. Selvin Single-molecule high-resolution imaging with photobleaching PNAS, April 27, 2004; 101(17): 6462 - 6465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Giacalone, S. Delobette, V. Gibaja, L. Ni, Y. Skiadas, R. Qi, J. Edington, Z. Lai, D. Gebauer, H. Zhao, et al. Optical Mapping of BAC Clones from the Human Y Chromosome DAZ Locus Genome Res., September 1, 2000; 10(9): 1421 - 1429. [Abstract] [Full Text]
|
|
|
|
|
|
J. Jing, Z. Lai, C. Aston, J. Lin, D. J. Carucci, M. J. Gardner, B. Mishra, T. S. Anantharaman, H. Tettelin, L. M. Cummings, et al. Optical Mapping of Plasmodium falciparum Chromosome 2 Genome Res., February 1, 1999; 9(2): 175 - 181. [Abstract] [Full Text]
|
|
