2.1.3 PCR amplification
The sequences of primers covering the 13 exons and flanking introns of
the PAH gene are shown in Supplement 1. PCR was performed in a total
volume of 25 μL, including 1 U Taq Plus DNA polymerase, 1× Taq Plus PCR
buffer, 2 pmol/L upstream and downstream primers, 2.5 mmol/L dNTPs, and
50 ng DNA template. The following cycling conditions were used for PCR:
94°C pre-denaturation for 15 min; 11 cycles of 94°C for 45 s, 62°C for
45 s (0.5°C drop per cycle), and 72°C for 1 min; 24 cycles of 94°C for
45 s, 57°C for 45 s, and 72°C for 1 min; and a final step of 72°C for 10
min. The amplified products were detected by 1% agarose gel
electrophoresis.