As also shown in the study, the risk of cross hybridization when using an nt analog like dInosine is minimal, since dInosine is sensitive to a mismatch in the position next to it and >5 mmoi will reduce hybridization. On the other hand, one should be aware that if an unintended target has many mismatches covered by dInosine and only a limited number of mmoi (<5 mmoi), this could lead to cross hybridization and false positivity. Furthermore, the assumption that sm all differ at the third codon base is an oversimplification. Some synonymous codons also differ at the first and second bases. Thus, even if the Ino18 probe could hybridize when 11 of 17 trimers were intact, with perfect matches at bases 1 and 2, the tolerance to mmoi of a highly dInosine-substituted probe like Ino18 is limited. Around half of its six surplus trimers must be reserved for sm occurring at codon positions 1 and 2. This leaves three trimers available for non-synonymous mismatches. However, a long probe is more likely than a short probe to have matches not neighboring mismatches.