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


Evans et al. BMC Clinical Pathology 2005 5:10   doi:10.1186/1472-6890-5-10

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