For the First round, the 30 ml reaction volume contained 3 ml DNA, 25 pmol/ml of each primer (pGp1 and pGp2), 3 ml of 106 PCR buffer (containing 1.5 mM magnesium chloride), 6.0 ml Qsolution, 0.2 mM deoxynucleotide triphosphates (dNTPs) and 1.0 U HotStar Taq polymerase (QIAGEN).