Metodology

1.PRA

1.1. DNA Extraction

1.2. PCR and restriction enzyme digestion and analysis

A 10-ng amount of purified mycobacterial DNA or 2 ml of supernatant collected form DNA extraction, were added to 50 ml PCR reaction mixture containing 50 mM KCl (ph 8.3); 1.5mM MgCl2; 10% glycerol; 200 mM of each dNTP; 0.5 mM of the primers Tb11 and Tb12 and 1.25 U of Taq polymerase. The amplification procedure was modified from an earlier protocol (Telenti et al., 1993) and consisted of 45 cyclesof 1 min at 94°C; 1 min at 65°C and 1 min at 72°C, followed by a final extesion step at 72°C for 7 min. A 10-ml volume of the PCR reaction mixture were analyzed by gel electrophoresis and staining with ethidium bromide and when positive, 15 ml was digested with 10 U BspI (shimozer of HaeIII, produced in our laboratory) or 6 U BstEII under mineral oil. Analysis of restriction enzyme products was done by gel electrophoreses on a 5% NuSieve GTG agarose gel, using a commercial 50 or 25bp DNA ladder as molecular weight marker.

1.3.Computer Analysis

Besides visual analysis of the digest, DNA patterns were submitted to computer analysis. Electrophoresis and photograph conditions were standardized to assure reproducibility within and between gels. Photographs were scanned and the images introduced into the GELCOMPAR software. Normalization was performed using two external reference markers per gel and a library of DNA patterns of reference strains was contructed.