## 2 Materials and Methods
### DNA Extraction and Sequencing  
The PstS-1 (Rv0934) and Ag85B (Rv1886c) coding genes in _M. microti_ and _M. tuberculosis_ were amplified by a polymerase chain reaction (PCR) and submitted to sequencing. Initially, _M. microti_ and _M. tuberculosis_ were grown in BD Difco(tm) Middlebrook 7H10 agar and incubated at 37 \({}^{\circ}\)C for 15 days. After the appearance of colonies, a single colony of each strain was taken and inoculated into 100 mL of Middlebrook 7H9 broth base supplemented with 0.05% Tween 80 and 10% ADC (Albumin-dextrose-catalase) in flasks, allowed to grow at 37 \({}^{\circ}\)C and shaken at 150 rpm until an A.U. of 0.6 (O.D. 600 nm) was achieved. The cells were harvested using centrifugation at 10,000 rpm for 15 min at 4 \({}^{\circ}\)C. The supernatant was discarded, and the pellet resuspended in 10 mL of glycerol 10%. The genomic DNA was extracted using the lysozyme and proteinase K method [58]. The DNA was resuspended in 50 \(\upmu\)L of distilled H\({}_{2}\)O and stored at \(-20\)\({}^{\circ}\)C for further use. Specific primers were designed for each gene based on the genome of _M. tuberculosis_ H37 Rv (GeneBank: AL123456.3). For PstS-1 (Rv0934), the forward primer 5\({}^{\prime}\)-ATAGTACGAGATCCTGAAAAATCGTTTGCC-3\({}^{\prime}\) and the reverse primer 5\({}^{\prime}\)-ATAGTACGAGAATTCCTAGCCTGAAAATCGT-3\({}^{\prime}\) were used with the restriction sites BamHI and EcoRI (underlined). For Ag85B (Rv1886c), the forward primer 5\({}^{\prime}\)-GGCTGAAATTCATGACAGACGTGACCCAAA-3\({}^{\prime}\) and the reverse primer 5\({}^{\prime}\)-CGCGAAAGCTICAGCCGGCCCTAACGAAC-3\({}^{\prime}\) were used with the restriction sites EcoRI and HindIII (underlined). The PCR conditions for PstS-1 (Rv0934) were used as follows: one initial denaturation cycle at 94 \({}^{\circ}\)C followed by 30 cycles of 1 min at 94 \({}^{\circ}\)C for denaturation, 1 min at 56 \({}^{\circ}\)C for annealing and 1 min at 72 \({}^{\circ}\)C for elongation. For Ag85B (Rv1886c), there was an initial denaturation cycle at 94 \({}^{\circ}\)C and then 35 cycles of 1 min at 94 \({}^{\circ}\)C for denaturation, 1 min at 55 \({}^{\circ}\)C for annealing and 1 min at 72 \({}^{\circ}\)C for elongation. The PCR amplicons were visualized with a 2% gel electrophoresis and stained with ethidium bromide. They were then cleaned using the Wizard(r) SV Gel and the PCR Clean-Up System and subcloned using a TOPOT(tm) cloning kit with pCR(tm)2.1 according to the manufacturer's instructions. Subsequently, the resulting vector constructs were sequenced using commercially available primers: M13 in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems(tm), Foster City, CA, USA) at the Biology Institute, UNAM. The sequences were assembled using the SnapGene(r) viewer 6.1 bioinformatics software (Dotmatics, Boston, MA, USA) and analyzed using the Basic Local Alignment Search Tool (BLAST; NCBI, Bethesda, MD, USA) and the Clustal Omega [59] bioinformatics tools. The constructed vectors of _M. microti_-TOPO-PstS-1, _M. microti_-TOPO-Ag85B, _M. tuberculosis_-TOPO-PstS1 and _M. tuberculosis_-TOPO-Ag85B were transformed into the competent _E. coli_ DH5\(\alpha\) by heat shock using standard procedures [60] and stored at \(-20\)\({}^{\circ}\)C for further use.