Pulse Field Gel Electrophoresis for Comparative Studies of Bacteria Diversity

The technique of pulse-field gel electrophoresis (PFGE) makes use of restriction enzymes that cleave DNA at specific restriction sites, generating DNA fragments of different sizes. These fragments can be separated according to size by agarose-gel electrophoresis and visualized using DNA stains (e.g. ethidium bromide), giving a characteristic banding pattern. Given the high specificity of restriction enzymes, the banding pattern generated is specific to each DNA sequence. This technique is, therefore, widely used by microbial epidemiologists to differentiate between bacterial strains. In this study, an effective assay for the PFGE method was established for the potential analysis of the diversity within environmental nitrifying bacterial cultures.. To this end the E. coli strain MG1655 was used to test different parameters including: grade of agarose, cellular concentration, enzyme concentration, and wash volumes. The importance of each parameter is noted in this report. Restriction analysis computer software tools were used to predict banding patterns for the restriction digests. The banding patterns predicted in silico strongly correlate to those obtained empirically in this study. Moreover the banding pattern in our experiment was also remarkably similar to that one reported by Tenover et al for an identical E. coli strain (2). Although an effective assay was established in the last experiment of this study, reproducibility and importance of these parameters and additional, potential relevant parameters are yet to be determined. _____________________________________________________________