The aim of these studies was to improve understanding of the design of probes to be used in a TMAC buffer system, by investigating variability and the inclusion of dInosines, other universal bases and wobbles. Specifically, we examined (i) variation (i.e. mismatch) tolerance, (ii) sensitivity to different mismatch distributions, (iii) utilization of dInosine as an nt analog, and (iv) specificity; we also present a new algorithm for prediction of hybridization results. In additional experiments, the question of the use of degeneracy versus a universal base was addressed. Furthermore, we investigated the use of the derived design criteria for detection of rotavirus RNA in clinical samples.