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Background  Large genomes contain families of highly similar genes that cannot be individually identified by microarray probes. This limitation is due to thermodynamic restrictions and cannot be resolved by any computational method. Since gene annotations are updated more frequently than microarrays, another common issue facing microarray users is that existing microarrays must be routinely reanalyzed to determine probes that are still useful with respect to the updated annotations.    Results   PICKY  2.0 can design shared probes for sets of genes that cannot be individually identified using unique probes.  PICKY  2.0 uses novel algorithms to track sharable regions among genes and to strictly distinguish them from other highly similar but nontarget regions during thermodynamic comparisons. Therefore,  PICKY  does not sacrifice the quality of shared probes when choosing them. The latest  PICKY  2.1 includes the new capability to reanalyze existing microarray probes against updated gene sets to determine probes that are still valid to use. In addition, more precise nonlinear salt effect estimates and other improvements are added, making  PICKY  2.1 more versatile to microarray users.    Conclusions  Shared probes allow expressed gene family members to be detected; this capability is generally more desirable than not knowing anything about these genes. Shared probes also enable the design of cross-genome microarrays, which facilitate multiple species identification in environmental samples. The new nonlinear salt effect calculation significantly increases the precision of probes at a lower buffer salt concentration, and the probe reanalysis function improves existing microarray result interpretations.

Shared probe design and existing microarray reanalysis using PICKY