one cycle at 94 ◦C for 20 s (denaturation), at 65 ◦C for 30 s (annealing),
and at 72 ◦C for 40 s (elongation); two cycles at 94 ◦C for 20 s
(denaturation); at 62 ◦C for 30 s (annealing); 72 ◦C for 40 s (elongation);
three cycles at 94 ◦C for 20 s (denaturation); at 59 ◦C for 30 s
(annealing); at 72 ◦C for 40 s (elongation); five cycles at 94 ◦C for 20 s
(denaturation); at 57 ◦C for 30 s (annealing); at 72 ◦C for 40 s (elongation);
twenty four cycles at 94 ◦C for 20 s (denaturation); at 55 ◦C
for 30 s (annealing); at 72 ◦C for 40 s (elongation); and then, final
extension at 72 ◦C for 10 min in a PTC-200 Peltier Thermal Cycler
(MJ Research, Watertown, MA)