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Nucleic Acids Research Advance Access published online on October 18, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm865
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© 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.

Chemistry

DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison

Daniel J. Fish1,*, M. Todd Horne1,2, Greg P. Brewood1, Jim P. Goodarzi1, Saba Alemayehu1, Ashwini Bhandiwad1, Robert P. Searles3 and Albert S. Benight1,2

1Portland Bioscience, Inc., 2Departments of Chemistry and Physics, Portland State University, Portland, 3Spotted Microarray Core, Oregon Health and Science University, Beaverton, OR, USA

*To whom correspondence should be addressed. Tel: (503) 725-2350; Fax: (503) 725-2305; Email: djf{at}pdxbio.com

Received June 26, 2007. Revised August 20, 2007. Accepted September 14, 2007.

Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters {Delta}H°, {Delta}S° and {Delta}G° of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to observed deviations from linearity. A detailed comparison of measured thermodynamic parameters with those calculated using the nearest-neighbor model was performed. Analysis revealed the nearest-neighbor model generally predicts mismatch duplexes to be less stable than experimentally observed. Results also show the relative stability of a tandem mismatch is highly dependent on the identity of the flanking Watson–Crick (w/c) base pairs. Thus, specifying the stability contribution of a tandem mismatch requires consideration of the sequence identity of at least four base pair units (tandem mismatch and flanking w/c base pairs). These observations underscore the need for rigorous evaluation of thermodynamic parameters describing tandem mismatch stability.


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