Thermodynamic parameters of duplex formation were derived by the van't Hoff analysis methods. The shape of each melting curve was fitted to the two-state model with linear base lines (22) using a nonlinear least-square program (23) . Unless otherwise stated, T m of DNA duplexes were measured at a concentration of 5 · 10 À7 M in 1· PCR buffer containing 10 mM Tris-HCl (pH 8.5), 5 mM MgCl 2 and 40 mM NaCl, as described earlier (23) .