In pursuit of this level of understanding, and in order to obtain reliable hybridization data, we chose to use the Luminex suspension array system in our studies. The inherent ability of the system to report the median of a high number of measurements (i.e. measuring hybridizations signals from 100 different beads) provides highly reliable data. Moreover, hybridization equilibrium is reached more rapidly using the suspension array system (taking around 15 min) than by solid phase hybridizations such as micro arrays (often overnight). A probe length of 70 nt was selected for our studies because of the elevated mismatch tolerance of this length compared with shorter probes (29) . However, the advantage of the extended length of the probe could possibly be countered by a loss of specificity. Hybridization studies using long (50 or more nt) probes in a 3 M TMAC buffer system have not been reported previously; in reports using other hybridization systems, however, it has been suggested that 50-mer or 70-mer probes should contain no more than 15-20 contiguous nt complementary to non-targets (29, 30) . Previously, the hybridization properties of long probes have been analyzed using microarrays, with overnight (16) and ConSortß, to find suitable regions for primers to be used in reverse-transcription PCR and a conserved region for a detection probe. The length of the black bars represents the frequency of variation as an average percentage conservation at each nt position (y-axis). The figure shows the alignment and variation of 214 Rotavirus A sequences in the nt position of the probes displayed in Figure 5C . ConSortß was used to group the variations of the probe region into two haplotypes, which were then used to construct the probes shown in Figure 5C and Supplementary Table S4A Letowski et al. found that, under microarray conditions, mismatches grouped at the 5 0 or 3 0 end of a 50-mer probe affected the binding to a target less than if the mismatches were distributed throughout or centered in the probe. Furthermore, the 50-mer probes with mismatches distributed along the whole probe were more destabilized than the probes that had mismatches centered in the duplex (34) . When Deng et al. studied mismatches in 50-mer microarray probes with 1-7 pm in different distribution patterns, they concluded that the signal intensity was decreased more by evenly distributed than by randomly distributed pm (32) . When 60-mer Figure 2A , 3A and 4A were plotted against the predicted ÁG calculated in Visual OMP TM 7.0 (DNA Software). The predicted ÁG was obtained for the interaction between probes and targets in 3M TMAC buffer at a hybridization temperature of 45 C. The percentage MFI is the MFI signal of a probe hybridized with a study target divided by the MFI signal of the same probe hybridized with its perfectly matching target (e.g. InflA probe against InflA target) at the same temperature. Regression lines were calculated using the SigmaPlot dynamic curve fitting system. A five parameter sigmoidal function gave the highest correlation [f = y 0 +a/ (1+exp (-(xx 0 ) oligonucleotides were hybridized in a microarray, mismatches located near the middle of the probe resulted in a greater reduction of signal intensity than those located at the ends (33) . Additionally, microarray experiments with short oligonucleotides (16-40 nt) and one mismatch or a nt insertion in all positions show that the hybridization signal decreases when the mismatching portion is centered (35, 36) . The matching segments are shorter when mismatches are centered than when they are located peripherally. Our results, using microsphere-bound 70-mer probes and Luminex technology in a buffer containing 3 M TMAC, confirm that the distribution of the mismatches is of great importance and that hybridization is stronger when there are a few longer uninterrupted sequences than when there are many short sequences. These effects are formulated in the NucZip algorithm.