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Applied and Environmental Microbiology, July 2005, p. 3753-3760, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3753-3760.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Empirical Establishment of Oligonucleotide Probe Design Criteria{dagger}

Zhili He,1 Liyou Wu,1 Xingyuan Li,2 Matthew W. Fields,3 and Jizhong Zhou1*

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831,1 Harbin Institute of Technology, Harbin, Heilongjiang, China 150001,2 Department of Microbiology, Miami University, Oxford, Ohio 450563

Received 16 September 2004/ Accepted 6 January 2005

Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of ≤85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (≤15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than –30 kcal/mol for 50-mer probes or –40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.

* Corresponding author. Mailing address: Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6038. Phone: (865) 576-7544. Fax: (865) 576-8646. E-mail: zhouj{at}ornl.gov.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, July 2005, p. 3753-3760, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3753-3760.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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