Nucleic Acids Research Advance Access originally published online on April 11, 2007
Nucleic Acids Research 2007 35(9):e70; doi:10.1093/nar/gkm154
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Nucleic Acids Research, 2007, Vol. 35, No. 9 e70
© 2007 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.
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Revision of the nonequilibrium thermal dissociation and stringent washing approaches for identification of mixed nucleic acid targets by microarrays
1Gulf Coast Research Laboratory, University of Southern Mississippi, 703 East Beach Dr, Oceans Springs MS 39564, USA, 2Center for Microbial Ecology, Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA and 3201 More Hall, Civil and Environmental Engineering, University of Washington, Seattle, WA 98195, USA
*To whom correspondence should be addressed. Tel: +1-206-685-7583; Fax: +1-206-685-3836; Email: panoble{at}washington.edu
Received August 16, 2006. Revised December 7, 2006. Accepted February 28, 2007.
Microarray experiments typically involve washing steps that remove hybridized nonspecific targets with the purpose of improving the signal-to-noise ratio. The quality of washing ultimately affects downstream analysis of the microarray and interpretation. The paucity of fundamental studies directed towards understanding the dissociation of mixed targets from microarrays makes the development of meaningful washing/dissociation protocols difficult. To fill the void, we examined activation energies and preexponential coefficients of 47 perfect match (PM) and double-mismatch (MM) duplex pairs to discover that there was no statistical difference between the kinetics of the PM and MM duplexes. Based on these findings, we evaluated the nonequilibrium thermal dissociation (NTD) approach, which has been used to identify specific microbial targets in mixed target samples. We found that the major premises for various washing protocols and the NTD approach might be seriously compromised because: (i) nonspecific duplexes do not always dissociate before specific ones, and (ii) the relationship between dissociation rates of the PM and MM duplexes depends on temperature and duplex sequence. Specifically for the NTD, we show that previously suggested use of reference curves, indices of curves and temperature ramps lead to erroneous conclusions.
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