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Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis

Parker, Richard C. and Watson, Robert M. and Vinograd, Jerome (1977) Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis. Proceedings of the National Academy of Sciences of the United States of America, 74 (3). pp. 851-855. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:PARpnas77

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Abstract

The cleavage of DNA by restriction endonucleases can be limited by the addition of ethidium bromide. When closed circular DNA is used as a substrate, DNA with one-site cleavages of one or both strands can be made by adding appropriate amounts of dye. The singly cleaved DNA is a complete set of full-length permuted linear molecules. Fractionation of the products of a digestion of the permuted linears with a single-hitting restriction endonuclease by gel electrophoresis yields a series of bands that can be used to determine relative molecular weights of the DNA fragments in the gel without the introduction of standards. It is possible to determine the relative molecular weight of a fragment to within ± 2.5%. These molecular weights immediately allow the determination of the HindIII and Hpa I maps of simian virus 40. The HindIII map of bacteriophage PM2 was determined by this method with one ambiguity that was resolved by using traditional techniques.


Item Type:Article
Additional Information:© 1977 by the National Academy of Sciences. Communicated by Norman Davidson, November 22, 1976. This research was done under the leadership of Prof. Jerome Vinograd. Without his contributions, direction, and inspiration this paper would not exist. While this manuscript was in its early stages of preparation his unfortunate death occurred. We thank Dr. J. Franklin for his advice about the mathematical aspects of this paper and M. Klymkowsky for his technical assistance. We thank Dr. N. Davidson for all of the help he gave us. This work was supported in part by National Institutes of Health Grants CA08014 and GM15327. This is Contribution no. 5417 from the Division of Chemistry and Chemical Engineering.
Subject Keywords:ethidium bromide; DNA of simian virus 40; DNA of bacteriophage PM2
Record Number:CaltechAUTHORS:PARpnas77
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:PARpnas77
Alternative URL:http://www.pnas.org/cgi/content/abstract/74/3/851
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9646
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Feb 2008
Last Modified:26 Dec 2012 09:50

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