In conclusion, we have demonstrated that the distribution of mismatches greatly affects probe hybridization. A minimum number of continuous, perfectly matching stretches (nucleation sites) is needed to initiate hybridization. Thus, if the target contains many variations and no long uninterrupted matching segments, use of a dInosine-containing probe, which partially overcomes the obstacles caused by mismatches, will be beneficial. With respect to hybridization prediction algorithms, a simple statement of percentage mismatch, as used in many such algorithms, does not adequately reflect the hybridization properties of a long duplex. The insertion of dInosines in the positions of variation could detect target nucleic acid that a consensus probe would fail to catch. Hence, while high dInosine content in a probe decreases the probe's binding capacity, it also covers mismatches, as required when creating mismatch-tolerant, broaddetection probes against highly variable target nucleic acid sequences such as those seen in RNA viruses. The dInosine probes can be made even more forgiving by using a lower hybridization temperature. Furthermore, the probability of cross hybridization is low because the mmoi neighboring the dInosines have a destabilizing effect on hybridization. While the aim of this study was to improve our understanding of variability and the effects of dInosines, other universal bases and wobbles in the design of probes to be used in a 3 M TMAC buffer system, with more exploratory work the results may also be relevant to other hybridization systems and could aid the development of hybridization-based diagnostic tools, including nanotechnological applications such as the volume-amplified magnetic nanobead detection assay (48, 49) .