A simple method for sizing large fragments of bacterial DNA separated by PFGE.

Abstract

Pulsed field gel electrophoresis (PFGE) of genomic DNA is a highly discriminatory molecular profiling technique for the epidemiological investigation of bacterial strains causing infections in human populations. It has been applied to study an increasing number of pathogenic bacterial species (Tenover et aL, 1995) and is particularly useful for Campylobacter jejuni (Owen et aL, 1995). The interpretation and comparison of PFGE profiles is simplified if the size of each individual DNA fragment is known. Various computer programs have been developed for sizing DNA fragments in conventional electrophoretic agarose gels and these are generally applicable to fragments in the range 100 bp— 30kbp but do not perform well on PFGE fragments in the range 40—900 kbp. This is because DNA migration in PFGE differs from that in conventional gels and a different relationship between mobility and size is required. The aim of the present study was to develop and evaluate a method for sizing DNA fragments in the range 40—900 kbp. We describe how to use the software package Microsoft Excel 5.0 to build a set of files designed to size DNA fragments for both conventional gels and PFGE.