**3.3 Mitochondrial cytochrome oxidase subunit 2 (COII)**

Polymorphism in mitochondrial cytochrome oxidase subunit 2 (COII) gene was analyzed using the Tcmit-10 and Tcmit-21 primers that amplified a fragment of approximately 375 bp [29]. The reaction was conducted in a final volume for each sample of 50 μl containing ~100 ng of DNA template, 1U Taq DNA polymerase (Thermo Fisher Scientific), 0.2mM of dNTPs, 1.5mM MgCl2, buffer 1X (10mM Tris-HCl pH 8.8, 50mM KCl, 0.8% Nonidet P40), 10 pmol of each primer (tcmit10 and tcmit21). The reaction was performed under the following temperature conditions: 95°C/5 min and 40 cycles of 95°C/45 sec, 48°C/45 sec and 72°C/1 min and a final extension of 72°C/10 min. The obtained fragments were digested with *Alu*I restriction endonuclease and the polymorphism was analyzed after electrophoresis in 6.5% acrilamide gel. Dm28c (TcI), Y (TcII), 3663 (TcIII), 4167 (TcIV), (TcV) and CL Brener (TcVI) strains were used as control.
