Field inversion gel electrophoresis in denaturing polyacrylamide gels

Abstract

The velocities of single stranded DNA molecules in denaturing polyacrylamide gels during symmetric and asymmetric field inversion were measured at different pulse times and gel concentrations. Under the conditions chosen in our study, pulse times as short as a few milliseconds lead to a retardation of DNA molecules larger than 400 bases. We found that a field inversion with an electric field in the forward direction of about double the strength of that applied in the backward direction is a good compromise between the degree of retardation, the temperature control requirements and the run time of the gel.

Separation of double-stranded and single-stranded DNA in polymer solutions: I. Mobility and separation mechanism

“…Figure 1 shows the dependence of the electrophoretic mobility on DNA size (dsDNA and ssDNA) at an electric field strength of 210 V/cm and at polymer concentrations of 1%, 2%, 5% and 10% in ªshort chainº, ªmedium chainº and ªlong chainº pDMA (with ªlong chainº pDMA, we did not perform experiments at 10%, due to the rather high viscosity, > 10 000 mm 2 /s). In all cases, we can observe the usual sigmoidal curve on the double logarithmic mobility vs. size plot, in agreement with separation of dsDNA in agarose gels [26,32], ssDNA in polyacrylamide gels [33] or dsDNA in entangled polymer solutions [17,28].…”

“…3 of [26]) and also for ssDNA in polyacrylamide gels (see Fig. 2 in [33]), which can now be explained by the most recent version of the BRF [25]. However, for DNA separation in polymer solutions, it still cannot account for the pore size dependence.…”

Finding a universal low viscosity polymer for DNA separation (II)

“…Above a size of about 70 bp, we can observe the usual sigmoidal curve on the double logarithmic molecules has hardly been studied in detail to date, as in usual agarose gels, they are not separated and also only recently, intercalators with strong fluorescence and strong binding became commercially available, making it possible to visualize those short molecules without the need to overload the gel or the capillary. Figure 7a also shows that for the small molecules (8-70 bp, regime I) the mobility does not continue to level-off as it is described in numerous publications (e.g., [38,[53][54][55][56][57][58] for separation in gels and [15, 281 for separation in polymer solutions), but that it increases with decreasing DNA size. This effect, which has been verified with different DNA standards (to exclude effects of repeating sequences) is most pronounced in 10% pDMA.…”

Gel electrophoresis of an end‐labeled DNA I. Dynamics and trapping in constant fields

“…5,are in good qualitative agreement with experimental data (see Fig. 2 in [21]): we predict a mobilitywhich decreases sharply past a critical size, N*,, this size decreasing with increasing field. This leads to a mobility decreasing with increasing field for large enough chains, an unusual feature that is also apparent in experiments.…”

“…This crossover also corresponds to the appearance of a nonlinear dependence of the mobility on the electric field; therefore, some progress is expected of PFGE. Recent studies, however, suggest that the effect of field-pulsing on single-strand DNA around one kb is moderate [21]. Another approach, in which a bulky streptavidin molecule is attached to one end of the DNA, was recently proposed by Ulanovsky et al [22].…”