2.4 Complete genome sequence amplification
For the two primer sets (Q1F/Q1R and Q2F/Q2R), PCR protocol was carried
out in 25μL of the reaction mixture: 12.5 μL of 2Phanta Max Buffer, 2μL
of sample DNA, 1μL of Phanta Max Super, 0.5 μL of dNTP Mix, 0.5 μL of
each primer (50 μM), and 8μL of ddH2O. The PCR
parameters were as follows: pre-denaturation at 94°C for 3 min; followed
by 35 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 15
s, extension at 72°C for 2min, and then followed by a final extension
step at 72°C for 10 min. For the Q3F/Q3R primer sets, PCR protocol was
carried out in 25μL of the reaction mixture: 12.5 μL of 2Es Taq Master
Mix (Dye), 3μL of sample DNA, 0.5 μL of each primer (50 μM), and 8.5μL
of ddH2O. The PCR parameters were as follows:
pre-denaturation at 95°C for 5min; followed by 35 cycles of denaturation
at 95°C for 45s, annealing at 60°C for 45s, extension at 72°C for 2min,
and then followed by a final extension step at 72°C for 10 min. The
purified
products were cloned into the pMD18-T Vector (TaKaRa) for sequencing,
and all sequencing reactions were performed in duplicate.