Table 1.

Details of the PCR protocols tested. All protocols included substitution of dTTP with dUTP and commenced with a uracil N-glycosylase (UNG) incubation step (37°C for 30 minutes) followed by a HotStarTaq DNA Polymerase (Qiagen) activation/UNG inactivation step (95°C, 15 min).

Protocol	Denaturation	Touchdown annealing cycles	Additional annealing cycles	Extension	Final extension
GP5+/GP6+	1 min, 94°C	-	2 min, 40°C (40–50 cycles)	1.5 min, 72°C	4 min, 72°C
TDP1	1 min, 94°C	2 min, 45°C to 40°C in 0.5°C decrements (11 cycles)	2 min, 40°C (29–39 cycles)	1.5 min, 72°C	4 min, 72°C
TDP2	1 min, 94°C	2 min, 50°C to 40°C in 1.0°C decrements (11 cycles)	2 min, 40°C (29–39 cycles)	1.5 min, 72°C	4 min, 72°C
TDP3	1 min, 94°C	2 min, 50°C to 40°C in 0.5°C decrements (21 cycles)	2 min, 40°C (19–29 cycles)	1.5 min, 72°C	4 min, 72°C
TDP4	1 min, 94°C	2 min, 55°C to 40°C in 1.0°C decrements (16 cycles)	2 min, 40°C (24–34 cycles)	1.5 min, 72°C	4 min, 72°C