Pulsed-Field Gel Electrophoresis of MRSA

Abstract

Pulsed-field gel electrophoresis (PFGE) is a genetic typing method that is widely used as a molecular epidemiological tool for studying the genetic diversity of Staphylococcus aureus and numerous other bacterial pathogens. For PFGE, intact bacterial cells are embedded in soft agarose plugs followed by lysis of the cell wall in situ to minimize shearing of the chromosome. The genome, which for S. aureus is approx 2.8 Mb, is then digested with a rare cutting restriction endonuclease and separated by agarose gel electrophoresis. The restriction fragments generated are too large to be resolved by conventional electrophoresis. Therefore, resolution of the bands is achieved in a “contour-clamped homogeneous electrical field“ where electrical current to the gel switches direction between multiple electrodes over a period of time. Initially, current switches are short (pulsed) but become longer (ramped) as electrophoresis continues. Banding patterns are captured by an imaging system and comparisons are made based on the Dice coefficient and the unweighted pair group method using arithmetic averages with BioNumerics software.