Nucleic Acids Research Advance Access originally published online on September 8, 2006
Nucleic Acids Research 2006 34(16):e111; doi:10.1093/nar/gkl642
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Nucleic Acids Research, 2006, Vol. 34, No. 16 e111
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Site-specific labeling of supercoiled DNA
Department of Pharmaceutical Sciences, College of Pharmacy University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA 1 Institute of Biosciences and Technology, Texas A&M University Health Sciences Center, 2121 West Holcombe Boulevard, Houston, TX 77030, USA
*To whom correspondence should be addressed. Tel: +1 402 559 1971; Fax: +1 402 559 9543; Email: ylyubchenko{at}unmc.edu
Received July 7, 2006. Revised August 16, 2006. Accepted August 16, 2006.
Visualization of site-specific labels in long linear or circular DNA allows unambiguous identification of various local DNA structures. Here we describe a novel and efficient approach to site-specific DNA labeling. The restriction enzyme SfiI binds to DNA but leaves it intact in the presence of calcium and therefore may serve as a protein label of 13 bp recognition sites. Since SfiI requires simultaneous interaction with two DNA recognition sites for stable binding, this requirement is satisfied by providing an isolated recognition site in the DNA target and an additional short DNA duplex also containing the recognition site. The SfiI/DNA complexes were visualized with AFM and the specificity of the labeling was confirmed by the length measurements. Using this approach, two sites in plasmid DNA were labeled in the presence of a large excess of the helper duplex to compete with the formation of looped structures of the intramolecular synaptic complex. We show that the labeling procedure does not interfere with the superhelical tension-driven formation of alternative DNA structures such as cruciforms. The complex is relatively stable at low and high pH (pH 5 and 9) making the developed approach attractive for use at conditions requiring the pH change.
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