Pulsed electrophoresis

Figure 27-36. Crossed field pulsed electrophoresis. (Courtesy Chrambach, A., Dunn, M.J., Radola, B.J., Eds. Advances in Electrophoresis Vol I VCH Press, Weinheim, Germany, 1987)...

One-dimensional pulsed electrophoresis (ODP) can size fractionate large DNA molecules. One experimental arrangement applies a relatively short electrical pulse followed by a much longer period of zero field strength (2,12). Presumably, during the field-on condition, molecular conformation is perturbed and relaxation then takes place during the field-off condition. As with TDP electrophoresis, mobility can be modulated with different pulse times. 

Many researchers have applied the reptational model to pulsed electrophoresis. Lumpkin et al. (9) have derived an expression for the time needed by a DNA molecule to reach a steady state orientation with an applied electrical field. This time can be related to the pulse time dependent mobilities seen in TDP by postulating a conformational transition from one orientation to another. As discussed by Lumpkin et al., minimum resolution should occur when molecules are all aligned. 

Figure 1. Pulsed Electrophoresis Equipment. Panel (A) depicts a PFG electrophoresis box with (a) being the space for the slab gel and...

The theoretical basis for pulsed electrophoresis is presently being constructed) and new associated phenomena are constantly being discovered. The lack of a firm physical understanding for this new electrophoretic phenomenon has not, however, prevented its successful application to many important biological problems. 

Some future developments should include applying pulsed electrophoresis to studies of very small DNA molecules, proteins, macromolecular complexes, cells and synthetic polymers. Fully understanding the physical mechanisms responsible for pulsed electrophoretic fractionation should enable researchers to extend resolution to even larger DNA molecules and fully generalize use of this new electrophoretic effect. 

The E dependence of R implied by Eqs. (24) and (25) introduces into the dynamic equations retarded field-dependent features which are at the heart of the complexity of conventional and pulsed electrophoresis of flexible chains. The model predicts two regimes which crossover for... 

As quoted by Slater et al. [125], the description sketched above is oversimplified. A more rigorous treatment [125, 128, 135, 150] predicts a non-monotonous M , dependence of the mobility around N, which has been confirmed experimentally [125,128] (Fig. 20). The asymptotic behavior in regimes i and ii, however, is not modified. The saturation of the mobility at large N is a dramatic nuisance for the electrophoretic separation of large biomolecules such as chromosomal DNA with sizes ranging from a few to 2 x 10 kilobase-pairs (kbp) (a base pair has a molecular weight around 360 Da). Until the recent introduction of pulsed electrophoresis, the practical range of separation hardly reached 100 kbp. 

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