“…Gel Electrophoresis and Hybridization. Fifty microliters from 100 µL of PCR products was precipitated on ice by adding 2 volumes of 100% ethyl alcohol, resuspended, applied on 1.8% agarose alkaline gel, 19 electrophoresed, and transferred overnight to nylon membranes (Hybond N, Bio Rad Laboratories, Hercules, CA). After drying and UV fixation, the membranes were prehybridized in a mixture containing 50% formamide, 5ϫ saline sodium citrate (SSC; 0.75 mol/L NaCl, 0.075 mol/L sodium citrate), 5ϫ Denhardt' s, 0.1% Ficoll (type 400; Pharmacia, Cologno Monzese, Italy), 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin (fraction V; Sigma), and 50 mmol/L NaHPO 4 (pH 7.0) at 42°C for at least 2 hours.…”