**3.4 Ribosomal RNA gene (24Sα)**

The study segment of the ribosomal RNA gene (DNAr24Sα) *T. cruzi* was performed using primers D71 and D72 [16]. These primers generate an amplification product of 110 bp and 125 bp for TcI to TcII, respectively. 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, 50 mM KCl, 0.8% Nonidet P40), 10 pmol of each primer (D71 and D72).

*Evaluation of Molecular Variability of Isolates of* Trypanosoma cruzi *in the State… DOI: http://dx.doi.org/10.5772/intechopen.104498*

Fragment amplification was carried out under the following temperature conditions: initial denaturation at 94°C/4 min and 30 cycles (94°C/1 min, 62.5°C/1 min, 72°C/1 min), followed by a final extension at 72°C/5 min. The visualization of the amplification product was performed by polyacrylamide gel electrophoresis in 7.5%. The gels were revealed by silver impregnation (DNA Silver Staining Kit/Amersham Biosciences). Dm28c (characterized as TcI) and CL Brener (characterized as TcII) strains were used as controls.

## **3.5 Spliced leader intergenic region (SL-IRac) gene**

The amplification of the spliced intergenic region (SL-IRac) gene was realized with TcIII and UTCC primers in order to distinguish TcIII (fragment of 200 bp) and other DTU (fragment of 150 to 157 bp). 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 (TcIII and UTCC). The reaction was performed under the following temperature conditions: 95°C/5 min, 3 cycles of 94°C/30 sec, touch down 70–64°C/30 sec, 72°C/1 min and 33 cycles of 94°C/30 sec, 62°C/30 sec and 72°C/1 min and final extension of 72°C/10 min. The final products were visualized by electrophoresis in 6.5% acrylamide gel. Dm28c and 3663 strains were used as control.
