PCR reactions were carried out in a final volume of 15 ml containing 0.4 mM of primer, 10mM of Tris–HCl (pH 8.3),
50mM of KCl, 2mM of MgCl2, 40ng of DNA, 100mM of each desoxyribonucleotide (dNTP) and 1.0 U of Taq polymerase (Invitrogen). Amplifications were performed in a thermal cycler (PTC-100MJ Research) programmed as 95 8C for 4 min, followed by 43 cycles of 1min at 95 8C, 1min at 35 8C and 1.5 min at 72 8C and a final extension at 72 8C for 7 min. Amplified fragmentswere separated by agarose (1.4%) gel electrophoresis stained by ethidium bromide and visualized under ultra-violet light. All PCR reactionswere heldtwice.