The
amplification reaction was performed with a volume of
25 ml, using the Takara Ex Taq DNA polymerase and buffer
system (Takara Mirus Bio Corporation). The final PCR
mixture comprised 16Ex Taq buffer (with 1.5 mM
MgCl2), 200 mM of each deoxynucleoside triphosphate,
0.2 mM of each primer, 1 unit Ex Taq DNA polymerase
and 50 ng template DNA. Amplification was carried out in
a thermocycler (GeneAmp PCR System 2400, PE
Biosystems) with the following cycling program: initial
denaturation at 94 uC for 5 min followed by 25 cycles of
94 uC for 10 s, annealing at 58 uC for 30 s, and extension at
72 uC for 2 min, and a final extension step at 72 uC for
10 min.