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Nucleic Acids Research 2006 34(4):e34; doi:10.1093/nar/gkl006
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Published online 1 March 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes

Matthew E. Pipkin1 and Mathias G. Lichtenheld1,2,3,*

1Department of Microbiology and Immunology, University of Miami, Miller School of Medicine Miami, FL, USA 2The Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine Miami, FL, USA 3The Center for HIV Research, University of Miami, Miller School of Medicine Miami, FL, USA

*To whom correspondence should be addressed at Mathias G. Lichtenheld, Department of Microbiology and Immunology, University of Miami Miller School of Medicine, 1580 N.W. 10th Avenue, Batchelor Children Research Institute Room 738, Miami, FL 33136, USA. Tel: +1 305 243 3301; Fax: +1 305 243 7211; Email: mlichten{at}med.miami.edu

Received January 6, 2006. Revised February 10, 2006. Accepted February 10, 2006.

The study of eukaryotic gene transcription depends on methods to discover distal cis-acting control sequences. Comparative bioinformatics is one powerful strategy to reveal these domains, but still requires conventional wet-bench techniques to elucidate their specificity and function. The DNase I hypersensitivity assay (DHA) is also a method to identify regulatory domains, but can also suggest their function. Technically however, the classical DHA is constrained to mapping gene loci in small increments of ~20 kb. This limitation hinders efficient and comprehensive analysis of distal gene regions. Here, we report an improved method termed mega-DHA that extends the range of existing DHAs to facilitate assaying intervals that approach 100 kb. We demonstrate its feasibility for efficient analysis of single-copy genes within a large and complex genome by assaying 230 kb of the human ADAMTS14-perforin-paladin gene cluster in four experiments. The results identify distinct networks of regulatory domains specific to expression of perforin and its two neighboring genes.


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