Recently, Majlessi et al. (15) studied the nucleation process during double helix formation of short probes, 18-28 nt, with RNA or DNA targets of varying lengths and number of mismatches, in a buffer containing lithium succinate and lithium lauryl sulfate at pH 5.1. Hybridization is initiated by random collisions, but occasionally the complex is stable enough to nucleate the hybridization process. After investigating their model, they suggested that one nucleation region of 9 nt is not enough for further zipping and formation of a double helix. Instead, the first nucleation site needs a second nucleation site so that they can then cooperatively induce the zipping mechanism. They reported that inactivation of one of the 9-nt sites reduced hybridization >2-fold. Interestingly, one complete turn of a dsDNA molecule consists of 10.4 nt (44) . It is conceivable that the first (often temporary) contact between two single nucleic acid strands (nucleation) should not exceed one turn of the dsDNA helix in length, to avoid torsional disturbance and faulty interlocking of the strands. From this point of view, the nucleation site should be long enough to minimize false contacts, and short enough to have minimal steric effects on the strands. Nucleation sites of 6-9 nt fulfil these criteria. The chance of two random single strands matching at a hexanucleotide is 1/16 394, and at a nonanucleotide is 1/ 1 048 576. A matching nonanucleotide will thus reduce the ratio of random successful to unsuccessful nucleations, i.e. those which do not lead to further hybridization in the subsequent zipping phase, a million-fold. The subject is far beyond the scope of this article; however, our data, using 70-mer probes in 3 M TMAC buffer, reveal that a target with two separate regions of 9 nt was enough for efficient hybridization when several shorter regions of 5-6 nt were available between mismatches during the hybridization process (InflA probe/26%9F, 2583 MFI). Increasing the number of mismatches, i.e. shortening the matching regions between the 9 nt flanking regions, caused failure of hybridization (InflA probe/33%9F, 133 MFI and InflA probe/74%9F target, 86 MFI). In contrast, the Ino18 probe, with dInosines covering the mismatches and nine matching nt at the 5 0 and 3 0 ends, hybridized strongly (Ino18/33%9F, 3894 MFI). Furthermore, the InflA probe/74%15nt or InflA probe/74%18nt combinations showed that one region of 15-18 nt was enough to induce and sustain hybridization reproducibly, even if the rest of the 70-mer probe contained 74% mismatches. Having two perfectly matching regions of 15 nt (15F) compared to one region of 15 nt (15 nt) gave 2.6-fold higher MFI for 33%15F compared to 33%15 nt and 4.5-fold higher MFI for 74%15F compared to 74%15 nt, at 45 C. The Ino24 probe, with no dInosine-free matching trimers, hybridized inefficiently with all targets (361-920 MFI at 45 C), showing that dInosine is relatively inefficient in creating nucleating regions. Thus, two nucleation sites of 9 nt are enough to cause hybridization in 3 M TMAC when they are placed next to each other to form a longer region or when there are enough shorter matching regions of 5-6 nt between them. Alternatively, dInosines could bridge the mismatches between the 9-nt regions.