After 5 min initial denaturation at 94ºC, the reaction mixture was run through 35 cycles (Techne Progene Thermal Cycler, USA) of denaturation for 30 sec at 94ºC, annealing for 30 sec at 55ºC, and extension for 30 sec at 72ºC, followed by an incubation for 15 min at 72ºC.