Figure 2

A multi-step laddered PCR Protocol. With this protocol, template DNA was amplified for 34 cycles with 5 minutes at 95°C for initial denaturation, 20 second at 94°C for denaturation, 30 second for annealing, 140 second at 68°C for extension and 10 minutes at 68°C for final extension. Annealing temperature was variable: it started from a relatively high temperature (55°C), and then decreased 1–2 degree each time until to 46°C. The temperature again increased 5 degree and stabilized at 51°C.