**3. Polymerase chain reaction (PCR)**

#### **3.1 Mini-exon gene**

The variability of the intergenic region of the gene of the mini-exon of the samples was studied using the technique of multiplex PCR, using primers TcI, TcII, Z3, Tr and ME [24]. These primers generate an amplification product of 200 bp (TcI), 250

bp (TcII), 150 bp (Z3) and 100 bp (*T. rangeli*). The reaction was conducted in a final volume for each sample of 50 μl containing ~100 ng of DNA template, 1U Taq Gold DNA polymerase (Invitrogen), 0.2mM of dNTPs, 1.5mM MgCl2, buffer 1X (10mM Tris-HCl pH 8.5), 10 pmol of each primer (TcI, TcII, Z3, Tr and ME). Fragment amplification was carried out under the following temperature conditions: initial denaturation at 94°C/5 min and 5 cycles (94°C/1 min, 50°C/1 min, 72°C/1 min), followed by 25 cycles (94°C/30 sec, 55°C/30 sec, 72°C/30 sec) and a final extension at 72°C/5 min. Amplification products were analyzed by electrophoresis on 2.5% agarose gel and visualized under UV light after ethidium bromide staining. Dm 28c (TcI), CL Brener (TcII), 3663 (Z3) and R1625 (*Trypanosoma rangeli*) strains were used as control.
