Next, we screened for potential structures that can form in the PM probes listed in Table 2 or their targets that could explain their poor hybridization specificity. To do this, we used Mfold (38) to calculate Gibbs free energies for intramolecular structures that can form in these PM probes and targets. Based on these Gibbs free energy values, we classified the probes and targets as having strong (S) [DG < (À3 kcal/mmol)], medium (M) [(À1 kcal/mmol) > DG > (À3 kcal/mmol)] and weak (W) [G > (À1 kcal/mmol)] potential for secondary structure. We found that several target and probe sequences could form substantial secondary structures, as displayed in Figure 6 . This could artificially lower the affinity of target to PM probes and thus lower the hybridization specificity. It is more difficult to model intermolecular structure in the solution-phase complex target and in the solidphase oligonucleotide probes. However, it appears likely that such structures could also have a similar negative impact on hybridization specificity.