Gene navigator system

Yeast chromosomes were prepared from 1 mL of yeast cells pre-grown in YP-20 g/L glucose medium and collected at the stationary phase of growth. Cells were treated with Zymolyase 20T and proteinase K in low-melting-point agarose blocks, transferred to a 1% agarose gel, and pulsed-field gel electrophoresis (PFGE) was performed at 8 °C using a Gene Navigator system (Amersham Pharmacia Biotech do Brasil Ltd.a., São Paulo, SP, Brazil) as previously described [29 (link),40 (link),41 (link),44 (link)]. Following electrophoresis, the gel was stained with ethidium bromide and photographed (Gel Doc™ XR, BioRad Laboratories, Hercules, CA, USA). The chromosomes separated by PFGE were transferred to a nylon membrane (Hybond-N⁺, GE Healthcare, Barueri, SP, Brazil) by capillary blotting. Labeling of DNA probes (see below), pre-hybridization, hybridization, stringency washes, and chemiluminescent signal generation and detection were performed with an AlkPhos kit (GE Healthcare) as recommended by the manufacturer. After hybridization, an autoradiography film (Hiperfilm™ ECL, Kodak, GE Healthcare) was exposed to the membrane for 1 to 2 h before it was developed. Images were obtained with Image Lab Software (Gel Doc™ XR) and annotated with Microsoft PowerPoint. Probes were generated by PCR using DNA from strain S288C as template with primers (Table 2) SUC100-F and SUC1320-R for the SUC2 gene.

The PFGE separation was performed according to Feitosa et al. [21] (link), with minor modifications. Briefly, DNA plugs were loaded onto the PFGE gel (0.8%), and chromosomal separation was carried out in the Gene Navigator System (Amersham Pharmacia Biotech, USA) at a constant temperature of 10°C. The best chromosomal band resolution was achieved in 1× TAE buffer with homogeneous pulses and interpolation for 168 h at 42 V (phase 1: pulse time 900 s/24 h; phase 2∶1800 s/24 h; phase 3∶2700 s/48 h; phase 4∶3600 s/48 h; phase 5∶4500 s/24 h). Schizosaccharomyces pombe chromosomes (Bio-Rad, USA) were run in parallel as standard size markers. After electrophoresis, the gel was stained with 0.5 µL/mL ethidium bromide (0.5 µL/mL; EtBr) for 15 min, and washed with distilled water for 15 min. The digital images of the gel were acquired with a L-Pix Touch documentation system (Loccus Biotecnologia, Brazil).

Chromosomal DNA was isolated and fractionated via PFGE as described elsewhere [20] (link), [21] (link). Briefly, 1.1% agarose gels were prepared in 0.5X TBE (45 mM Tris; 45 mM boric acid; 1 mM EDTA, pH 8.3), and agarose plugs containing the samples were loaded into the gels and electrophoresed using the Gene Navigator System (Amersham Pharmacia Biotech) at 13°C for 132 hours. The gels were then stained with ethidium bromide (EtBr) (0.5 mg/mL). The chromosomal bands of T. rangeli (Choachí and SC-58 strains) and T. cruzi (CL Brener clone) were fractioned using a protocol optimized to separate small DNA molecules in the CHEF Mapper system to assess the presence of minichromosomes.