As expected, hybridization of the InflA probe to targets with 26% random mutations in a region between two flanking regions of 5 (26%5F), 7 (26%7F), 9 (26%9F) or 15 (26%15F) perfectly matching nt showed that shorter perfectly matching flanking regions reduced the MFI. All the 26% targets hybridized with the InflA probe at 45 C but, at 55 C, the 26%5F target failed to hybridize (5.6% of the total MFI, 271 MFI). Like the 33%12F and the 16-pm target, the 26%5F target contained 16 mutations (Figures 2A, B, 3A and B). The 33%12F target (45% of the total MFI, Figure 3A ), with its two regions of 12 uninterrupted nt, hybridized much more strongly than the 26%5F (27% of the total MFI) or 16-pm (6.1% of the total MFI) targets, which both contained several shorter matching regions, of 6, 5, 4 and 3 nt ( Table 2) . The same effect, i.e. that a few long perfectly matching regions result in better hybridization than several shorter regions, was also seen with the three targets that had 14 mismatches: the 26%9F, the 33%15F and the 14-pm targets at both temperatures ( Table 2 ). The 74%9F target, with only 12 matching nt dispersed in the central region, did not hybridize to the InflA probe (1.4% of the total MFI, 86 MFI, 45 C; and 0.6% of the total MFI, 30 MFI, 55 C), while the 26%9F target, with 38 matching nt (three regions of 5 nt and one region of 6 nt), did hybridize (44% of the total MFI, 2583 MFI, 45 C; and 24% of the total MFI, 1125 MFI, 55 C). This shows that the two flanking regions of nine matching nt did not cause hybridization alone. Furthermore, the 74%12F, with 11 matching nt dispersed in the central These experiments, taken in conjunction with the InflA probe hybridizing to the 21-gm but not to the 21-pm target, confirm that both the number of mismatches and their distribution are important. It is reasonable to assume that perfectly matching sequences of a minimum length function as nucleation sites (15) which initiate hybridization between the probe and the target.