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Applied and Environmental Microbiology, May 2003, p. 2848-2856, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2848-2856.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays

Hidetoshi Urakawa,1,{dagger} Said El Fantroussi,1,{ddagger} Hauke Smidt,1,§ James C. Smoot,1 Erik H. Tribou,1 John J. Kelly,2 Peter A. Noble,1 and David A. Stahl1*

Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington 98195,1 Department of Biology, Loyola University Chicago, Chicago, Illinois 606262

Received 26 August 2002/ Accepted 2 January 2003

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

* Corresponding author. Mailing address: Civil and Environmental Engineering, University of Washington, 302 More Hall, Box 352700, Seattle, WA 98195. Phone: (206) 685-3464. Fax: (206) 685-9185. E-mail: dastahl{at}u.washington.edu.

{dagger} Present address: National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8606, Japan.

{ddagger} Present address: Unit of Bioengineering, University of Louvain, B-1348 Louvain-la-Neuve, Belgium.

§ Present address: Wageningen University, 6703 CT Wageningen, The Netherlands.

Applied and Environmental Microbiology, May 2003, p. 2848-2856, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2848-2856.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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