MOTIVATION: Target-specific hybridization depends on oligo-probe characteristics that improve hybridization specificity and minimize genome-wide cross-hybridization. Interplay between specific hybridization and genome-wide cross-hybridization has been insufficiently studied, despite its crucial role in efficient probe design and in data analysis. RESULTS: In this study, we defined hybridization specificity as a ratio between oligo target-specific hybridization and oligo genome-wide cross-hybridization. A microarray database, derived from the Genomic Comparison Hybridization (GCH) experiment and performed using the Affymetrix platform, contains two different types of probes. The first type of oligo-probes does not have a specific target on the genome and their hybridization signals are derived from genome-wide cross-hybridization alone. The second type includes oligonucleotides that have a specific target on the genomic DNA and their signals are derived from specific and cross-hybridization components combined together in a total signal. A comparative analysis of hybridization specificity of oligo-probes, as well as their nucleotide sequences and thermodynamic features was performed on the database. The comparison has revealed that hybridization specificity was negatively affected by low stability of the fully-paired oligo-target duplex, stable probe self-folding, G-rich content, including GGG motifs, low sequence complexity and nucleotide composition symmetry. CONCLUSION: Filtering out the probes with defined 'negative' characteristics significantly increases specific hybridization and dramatically decreasing genome-wide cross-hybridization. Selected oligo-probes have two times higher hybridization specificity on average, compared to the probes that were filtered from the analysis by applying suggested cutoff thresholds to the described parameters. A new approach for efficient oligo-probe design is described in our study. CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The SAMSON package is a tool for advanced analysis of primary DNA, RNA and protein structures. The package consists of 16 programs performing statistical analysis and comparison of biopolymer sequences, search for homologies, translation of DNA and RNA sequences into amino acid sequences, splicing of RNA sequences and restriction map construction, recognition of functionally related sites in biopolymer molecules, textual analysis of DNA and RNA regulatory sites and prediction of intermolecular hybridization sites in DNA and RNA molecules.