Pulse-field Gel Electrophoresis for Epidemiological Studies of Streptococcus pneumoniae

Abstract

Electrophoresis techniques have allowed for very effective separation of nucleic acids on the basis of molecular weight. Conventional DNA electrophoresis can only separate DNA fragments smaller than 50 kb. Above this size, DNA fragments are larger than the pore size of the matrix. They can travel through a gel matrix by deforming their shape, parallel to the field in order to pass through the pores. This mode of migration is called “reptation” and all large molecules migrate at the same rate. Shwartz et al. (1) were the first to introduce the concept that molecules larger than 50 kb can be separated by using two alternating electric fields, one homogeneous and the other nonhomogeneous. In pulsed field gel electrophoresis (PFGE) the electrical field is applied alternately in two directions. The orientation of DNA molecules is perturbed and they have to reorient to move efficiently in response to the new field. The time required for this reorientation has been found to be proportional to the molecular weight. Larger DNA molecules take more time to realign after the fields are switched than do smaller ones. Thus, the reorientation introduces a size dependence that is absent during simple “reptation.” The timing of the electrical field in each direction is called the pulse time: too fast or too slow and no separation occurs. However molecules of DNA, whose reorientation times are less than the period of the electric pulse, will be fractionated according