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