Two-dimensional pulsed field minigel electrophoresis for the Cryptococcus neoformans chromosomes characterization: bioinformatic procedure

Abstract

Cryptococcus neoformans is a pathogenic yeast that causes meningoencephalitis, specially in AIDS patients. Epidemiological studies of clinical isolates have been encouraged due to the increase in its incidence for infection monitoring and control. Pulsed Field Gel Electrophoresis (PFGE) is the most popular technique for microbial molecular subtyping with epidemiological purpose, because of its versatility and reproducibility, but the analysis of eukaryotic genomes, like C. neoformans, in a single PFGE experiment, does not reveal mutations that do not alter the chromosomes size. That is why the author proposes a two-dimensional PFGE methodology for the C. neoformans genome characterization. The present describes the bioinformatic procedure used to design and verify in silico the efficacy of such methodology. The resolution of the intact chromosomes was achieved in the first dimension (1D) after 9.3 h of electrophoresis in the CHEF minichamber, applying pulses between 30 and 90s. The chromosomal fingerprint was obtained in the second dimension (2D) after restrict the individual bands with Not I or Sfi I and then separate the fragments in the CHEF minichamber applying pulses between 1 and 15 s for 5 h. The proposed methodology reveals the genetic differences in the genome of C. neoformans strains that were invisible in 1D, in 14.3 h of electrophoresis (1D + 2D), in contrast with the 30-40 h that takes a PFGE in regular electrophoretic chambers. Such methodology could be applied for studying the genome of other unicellular eukaryotic species.