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Nucleic Acids Research 2006 34(3):e26; doi:10.1093/nar/gnj024
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Published online 13 February 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

On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes

Lukas M. Wick, Jean Marie Rouillard3, Thomas S. Whittam, Erdogan Gulari3, James M. Tiedje1 and Syed A. Hashsham1,2,*

National Food Safety and Toxicology Center, Michigan State University East Lansing, MI, USA 1Center for Microbial Ecology, Michigan State University East Lansing, MI, USA 2Department of Civil and Environmental Engineering, Michigan State University East Lansing, MI, USA 3Department of Chemical Engineering, University of Michigan Ann Arbor, MI, USA

*To whom correspondence should be addressed. Tel: +1 517 355 8241; Fax: +1 517 355 0250; Email: hashsham{at}egr.msu.edu

Received November 22, 2005. Revised January 26, 2006. Accepted January 26, 2006.

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (Td-w, temperature of maximum specific dissociation rate constant), obtained from probe–target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.


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