Improvement of oligonucleotide probe design criteria for functional gene microarrays in environmental applications

To optimize oligonucleotide probe design criteria, PCR products with different similarities to probes were hybridized to a functional gene microarray designed to detect homologous genes from different organisms. In contrast to more restrictive probe designs based on a single criterion, simultaneous consideration of the percent similarity (≤90%), the length of identical sequence stretches (≤20 bases), and the binding free energy (≥-35 kcal mol^-1) was found to be predictive of probe specificity.