Physical Mapping of the Human Genome by Pulsed Field Gel Electrophoresis

DNA fragments larger than 25 kb are not resolved effectively by standard gel electrophoresis. Pulsed field gel electrophoresis (PFGE), first described by Schwartz and Cantor in 1984 (1 ), is a technique that allows for the separation of large DNA fragments of up to several mega base pairs (mbp) in length by periodically alternating the direction of the electric field applied to the gel. One important aspect of PFGE is the near linearity of the separation. This feature results in high resolution and allows for the effective separation of small DNA fragments between 10–20 kb as well as much larger fragments in the 100–1000 kb size range. The resolution of both large and small DNA fragments by PFGE is thought to result from the differing abilities of DNA molecules of different lengths to reorientate to the changed field direction. The PFGE equipment originally developed by Schwartz and Cantor (1 ) applied pulsed alternating orthogonal electric fields to the gel and was able to resolve DNA fragments of up to 2 mbp. However, the electric field generated by this early equipment was nonuniform and caused a skewing of the DNA tracks toward the edge of the gel with concomitant loss of resolution. A number of alternative electrophoretic configurations have been developed in order to overcome these problems and to increase the maximum size limit of resolvable DNA fragments. These include the periodic inversion a single electric field (field-inversion gel electrophoresis, FIGE; 2 and see Chapter 13 ), rotating gels (Waltzer; 3 ), transverse fields applied to vertical gels (TAFE;4 ), and contour-clamped homogeneous electric fields (CHEF;5 ).