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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 (&lt; or =90%), the length of identical sequence stretches (&lt; or =20 bases), and the binding free energy (&gt; or =-35 kcal mol(-1)) was found to be predictive of probe specificity.

Improvement of Oligonucleotide Probe Design Criteria for Functional Gene Microarrays in Environmental Applications