Table 2.

Standard PCR and sequencing primers, primer combinations and annealing temperatures used

Region	Forward primer	Reverse primer	Annealing temperature
ex5-in6 (PCR 1)	5F2898: TTGACAGCCTYGATGATGAT	Svex7R3420: ATCTTGAAAATCCAGCCC	52°C
ex5-in6	5F3062: AATGATGASGGGAAGAATTTTGC	Svex7R3420	52°C
ex6-ex7 (PCR 2)	vex6F3263: GYCARCTYCTTGAGGCTGC	7R3883: ATVCCCATGCTGAAKAGCTCYTG	59°C
ex5-ex6	5F2898	vex6R3371: YMTCRACACTGGGAGTGGAG	54°C
ex5-ex6	5F3062	vex6R3371	57°C
ex6-in6	vex6F3263	Svex7R3420	52°C
ex7	vex7F3418: GGCTGGATTTTCAAGATGG	7R3883	55°C

PCR mix: 20 to 40 μl reactions; 0.2 mM dNTPs, 0.25 μM of each of the primers, 1× Phusion HF buffer, 0.008 U/μl Phusion polymerase. The PCR conditions were as follows: initial denaturation at 95°C for 30 s followed by 35 cycles of 95°C for 9 s, annealing at a temperature specified below for 30 s, and 72°C for 30 s. The PCR ended with 7:30 minutes at 72°C and subsequent soak at 10°C.