Polymerase chain reaction-Sanger sequencing
Each 25-µl PCR mixture was prepared as follows: 0.5 µl 5 U/µl D-Taq enzyme, 1 µl dNTP Mix (10 mM), 5 µl 5XPCR Buffer, 1 µl 2C19 * 2-F2 (10 µM), 1 µl 2C19 * 2-R1 (10 µM), 1 µl 2C19 * 3-F1 (10 µM), 1 µl 2C19 * 3-R2 (10 µM), 1 µl 2C19 * 17-F1 (10 µM). 1 µl 2C19 * 17-R1 (10 µM), mixture, 10.5 µl deionized water and 2 µl DNA template. Conventional PCR was carried out using the SLAN-96S instrument (Shanghai Hongshi) with denaturation at 95°C for 30 s, 45 cycles of 95°C for 2 s and 60°C for 5 s, followed by 72°C for 10 min.
In short, 2 µl DNA template was added to a D-Taq enzyme containing 0.5 µl 5 U/µl, 1 µl dNTP Mix (10 mM), 5 µl 5XPCR Buffer, 1 µl 2C19 * 2-F2 (10 µM), 1 µl 2C19 * 2-R1 (10 µM), 1 µl 2C19 * 3-F1 (10 µM), 1 µl 2C19 * 3-R2 (10 µM), 1 µl 2C19 * 17-F1 (10 µM), 1 µl 2C19 * 17-R1 (10 µM) and 10.5 µl in deionized water. The amplification conditions were as follows: denaturation at 95°C for 30 s, 45 cycles of 95°C for 2 s 60°C for 5 s, followed by 72°C for 10 min. The amplified products were sent for bidirectional Sanger sequencing.