PCR Product Sequencing Protocol

Before sequencing, the PCR products were purified using a Qiagen PCR purification kit (Qiagen) following manufacturer’s instruction. The cycle sequencing PCR was then performed by BigDye Chain Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) applying manufacturer’s instructions. The thermal cycling profile comprised (i) initial denaturation at 94°C for 1 minute, (ii) 25 cycles of denaturation at 94°C for 10 seconds, annealing at 58°C for 5 seconds and extension at 60°C for 4 minutes, and (iii) final extension at 60°C for 10 minutes. After cycle sequencing PCR, the products were purified using BigDye XTerminator® Purification Kit (Applied Biosystems). Then, sequencing of the purified cycle sequencing products was executed on the ABI PRISM 310 automated sequencer (Applied Biosystems, USA) [7 (link)].