Cation was carried out with 0.4 mM of each primer, 5 U of Pfu polymerase enzyme (Promega), 2.5 mM of dNTPs, and 100 ng of genomic T. cruzi DNA as template in 50 ml final reaction volume. The PCR cycle consisted of 30 rounds of 94uC for 45 s, 63uC for 45 s, and 72uC for 45 s, with a first step of 2 min at 94uC and one last step of 5 min at 72uC. PCR products were analyzed by electrophoresis in 2 agarose gel. Amplicons were purified and both strands sequenced with the primers used for amplification. Chromatograms were visually examined to determine the presence of C and/or T in the first position of the codon 342. Firstly TS-51/TS-31 primer set was used and those genomes rendering only the Tyr342 codon were then subjected to the other three PCR reactions. The IUPAC nomenclature for the genetic code was used to define single nucleotide polymorphism (SNP) positions with mixed base identification set to 15 of the highest peak. Sequences were deposited in the GenBank 
with the accession numbers KC286514, KC286515, KC286516, KC286517, KC286518, KC286519, KC286520, KC286521, KC286522, KC286523, KC286524, KC286525, KC286526, KC286527, KC286528, KC286529, KC286530, KC286531, KC286532, KC286533, KC286534, KC286535, KC286536, KC286537, KC286538, KC286539, KC286540, KC286541, KC286542, KC286543, KC286544, KC286545, KC286546, KC286547, KC286548, KC286549, KC294586 and KC294587.Results Quantification of aTS and iTS genes in the genome of T. cruziTo analyze the distribution of TS-encoding genes in the genome of parasites analyzed by Risso et al [37], we KS 176 performed a quantitative analysis of aTS and iTS by real-time PCR on DNA samples. The single copy Pvdh gene [41] was included as internal reference to standardize the number of haploid genome copies in the test. Primers that amplify the region containing the single nucleotide polymorphism (SNP) that determines the loss of enzymatic activity were used together with two probes that differ in only one base (T/C transition) and comprise the Tyr codon (to hybridize aTS genes) or His codon (complementary to the sequence of iTS genes), respectively. No cross-recognition between the aTS and iTS probes under test conditions was found, as assayed with plasmids harboring the corresponding gene. No Ct could be determined with the iTS-complementary probe for lowvirulence TcI strains, indicating no detection of iTS genes carrying the T/C transition. As shown in Table 1, the genome of these T. cruzi isolates harbors 28 to 32 copies of aTS-coding genes. On the other hand, data obtained from the aggressive strains RA, Cvd, CL Brener and the clone Q501/3 (all belonging to TcVI) and Br (TcII) indicated that both the aTS and iTS genes were present with high variability in the gene copy number (Table 1). In these genomes, the aTS/iTS rate was high for TcII (3/1) and quite balanced in TcVI (2/1 and 1/1).Sequence comparison of the region around the codonA 455-bp consensus sequence of each T. cruzi stock was obtained by comparison of forward and reverse sequences of the TS-51/TS-31 PCR reactions. Sequences from the different stocks were aligned and compared by using ClustalW2 program [42]. A clustering tree was built by using SplitsTree4 [43] with the following options: i) the “UncorrectedP” method which HIF-2��-IN-1 computes the proportion of positions at which two sequences differ was used; ii) the ambiguous state codes (such as W, M, V …) were handled with the option `Average’ meaning that the contribution at a site is averaged ove.Cation was carried out with 0.4 mM of each primer, 5 U of Pfu polymerase enzyme (Promega), 2.5 mM of dNTPs, and 100 ng of genomic T. cruzi DNA as template in 50 ml final reaction volume. The PCR cycle consisted of 30 rounds of 94uC for 45 s, 63uC for 45 s, and 72uC for 45 s, with a first step of 2 min at 94uC and one last step of 5 min at 72uC. PCR products were analyzed by electrophoresis in 2 agarose gel. Amplicons were purified and both strands sequenced with the primers used for amplification. Chromatograms were visually examined to determine the presence of C and/or T in the first position of the codon 342. Firstly TS-51/TS-31 primer set was used and those genomes rendering only the Tyr342 codon were then subjected to the other three PCR reactions. The IUPAC nomenclature for the genetic code was used to define single nucleotide polymorphism (SNP) positions with mixed base identification set to 15 of the highest peak. Sequences were deposited in the GenBank with the accession numbers KC286514, KC286515, KC286516, KC286517, KC286518, KC286519, KC286520, KC286521, KC286522, KC286523, KC286524, KC286525, KC286526, KC286527, KC286528, KC286529, KC286530, KC286531, KC286532, KC286533, KC286534, KC286535, KC286536, KC286537, KC286538, KC286539, KC286540, KC286541, KC286542, KC286543, KC286544, KC286545, KC286546, KC286547, KC286548, KC286549, KC294586 and KC294587.Results Quantification of aTS and iTS genes in the genome of T. cruziTo analyze the distribution of TS-encoding genes in the genome of parasites analyzed by Risso et al [37], we performed a quantitative analysis of aTS and iTS by real-time PCR on DNA samples. The single copy Pvdh gene [41] was included as internal reference to standardize the number of haploid genome copies in the test. Primers that amplify the region containing the single nucleotide polymorphism (SNP) that determines the loss of enzymatic activity were used together with two probes that differ in only one base (T/C transition) and comprise the Tyr codon (to hybridize aTS genes) or His codon (complementary to the sequence of iTS genes), respectively. No cross-recognition between the aTS and iTS probes under test conditions was found, as assayed with plasmids harboring the corresponding gene. No Ct could be determined with the iTS-complementary probe for lowvirulence TcI strains, indicating no detection of iTS genes carrying the T/C transition. As shown in Table 1, the 
genome of these T. cruzi isolates harbors 28 to 32 copies of aTS-coding genes. On the other hand, data obtained from the aggressive strains RA, Cvd, CL Brener and the clone Q501/3 (all belonging to TcVI) and Br (TcII) indicated that both the aTS and iTS genes were present with high variability in the gene copy number (Table 1). In these genomes, the aTS/iTS rate was high for TcII (3/1) and quite balanced in TcVI (2/1 and 1/1).Sequence comparison of the region around the codonA 455-bp consensus sequence of each T. cruzi stock was obtained by comparison of forward and reverse sequences of the TS-51/TS-31 PCR reactions. Sequences from the different stocks were aligned and compared by using ClustalW2 program [42]. A clustering tree was built by using SplitsTree4 [43] with the following options: i) the “UncorrectedP” method which computes the proportion of positions at which two sequences differ was used; ii) the ambiguous state codes (such as W, M, V …) were handled with the option `Average’ meaning that the contribution at a site is averaged ove.
