The PCR conditions were 94℃ for 5 min followed by 38 cycles
of 94℃ for 1 min, 35-62℃ (depending on annealing
temperature of the primer pair) for 1 min, then 72℃
for 2 min, and a final extension for 10 min at 72℃.
The amplification products were heat-denatured for
5 min, snap cooled on ice and then separated on 6%
polyacrylamide denaturing sequencing gel (PAGE),
silver stained for observation (Sambrook et al., 1989).