The semi-nested touchdown PCR produced a 613 bp fragment using the following conditions: initial denaturation at 95 for 5 min, followed by 5 cycles consisting of denaturation at 95 for 15 s, annealing at 67 for 15 s, and extension at 72 for 30 s, 10 cycles carried out under the same conditions (except for the annealing temperature at 66), and 20 cycles with annealing at 65, followed by a final extension at 72 for 8 min.