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Published online 2 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 2 681-690
© 2004 Oxford University Press

Guidelines for incorporating non-perfectly matched oligonucleotides into target-specific hybridization probes for a DNA microarray

Inhan Lee*, Alan A. Dombkowski1 and Brian D. Athey

Michigan Center for Biological Information and Department of Psychiatry, University of Michigan, 3600 Green Court, Suite 700, Ann Arbor, MI 48105 and 1 Institute of Environmental Health Science, Wayne State University, Detroit, MI 48201, USA

*To whom correspondence should be addressed. Tel: +1 734 615 5918; Fax: +1 734 998 8571; Email: inhan{at}umich.edu

Sequence-specific oligonucleotide probes play a crucial role in hybridization techniques including PCR, DNA microarray and RNA interference. Once the entire genome becomes the search space for target genes/genomic sequences, however, cross-hybridization to non-target sequences becomes a problem. Large gene families with significant similarity among family members, such as the P450s, are particularly problematic. Additionally, accurate single nucleotide polymorphism (SNP) detection depends on probes that can distinguish between nearly identical sequences. Conventional oligonucleotide probes that are perfectly matched to target genes/genomic sequences are often unsuitable in such cases. Carefully designed mismatches can be used to decrease cross-hybridization potential, but implementing all possible mismatch probes is impractical. Our study provides guidelines for designing non-perfectly matched DNA probes to target DNA sequences as desired throughout the genome. These guidelines are based on the analysis of hybridization data between perfectly matched and non-perfectly matched DNA sequences (single-point or double-point mutated) calculated in silico. Large changes in hybridization temperature predicted by these guidelines for non-matched oligonucleotides fit independent experimental data very well. Applying the guidelines to find oligonucleotide microarray probes for P450 genes, we confirmed the ability of our point mutation method to differentiate the individual genes in terms of thermodynamic calculations of hybridization and sequence similarity.


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