PCR was performed in a volume of 25 μl containing 21 μl of Platinum PCR SuperMix (Invitrogen), 200 nM of each primer, and 25 ng template DNA. Samples were amplified using the following cycling parameters: 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds. PCR products were assessed for quality and yield on the NanoDrop spectrophotometer (Thermo Scientific Waltham MA, USA) and by gel electrophoresis on a 2% agarose gel. The remaining 15 μl of PCR amplicons were treated with 5 units of shrimp alkaline phosphatase (SAP) and 2 units of exonuclease I (ExoI) for 1 hour at 37°C, followed by 15 minutes at 75°C to remove excess dNTPs and primers, respectively.
