Field inversion gel electrophoresis

Figure 27-33. Field Inversion Gel Electrophoresis of DNA Molecules from 15 To 200 kbp.

Zimm, B.H. Lakes-straits model of field-inversion gel electrophoresis of DNA, J. Chem. Phys., 94, 2187,1991. 

Clones are auxotrophically selected in top agar and screened for completeness by multiplex PCR. Those that amplify all PCR products are screened for size by restriction digestion and either pulsed-field or field inversion gel electrophoresis (PFGE or FIGE), followed if necessary by Southern blot. Clones can also be sequenced (6), although it is likely to be difficult to isolate significant qnantities of clone DNA separately from yeast chromosomal DNA. 

VII. 4 Conclusion In conclusion, the BRM predicts that field-inversion gel electrophoresis should improve the separation of "plateau molecules if two unequal fields are used. In this case, the reverse field, which is of lesser intensity, serves to reduce the orientation of the reptation tube since this is field-induced, this is much faster than the tube relaxation process which is the basis of the intermittent-field technique. Experimentally, this reptation induced effect has been observed, but a potentially even more powerful "resonance- ike effect has been observed to exist as well. This latter effect leads to band-inversion however, and since it occurs for pulse durations t =x (N)involves chain movements inside its tube and/or movements of only parts of the tube, which have not been discussed in this article. 

By extrapolating to zero field strength it is possible to find an inverse relationship between electrophoretic mobility and molecular weight (14). This relationship contrasts with that obtained with a Ferguson plot (15) normally used for proteins, but also frequently and often erroneously used for DNA. However, the inverse relationship is fully predicted by reptation theory (3, 9). In summary, determining precise DNA molecular weights using gel electrophoresis requires careful attention to experimental detail and Judicious choice of data workup. 

Electroosmosis is one of several electrokinetic effects that deal with phenomena associated with the relative motion of a charged solid and a solution. A related effect is the streaming potential that arises between two electrodes placed as in Figure 9.8.1 when a solution streams down the tube (essentially the inverse of the electroosmotic effect). Another is electrophoresis, where charged particles in a solution move in an electric field. These effects have been studied for a long time (37, 38). Electrophoresis is widely used for separations of proteins and DNA (gel electrophoresis) and many other substances (capillary electrophoresis). 

The BRM has been successful in explaining continuous-field gel electrophoresis data. In particular, band-inversion, the "plateau" mobility and the p 1/L law are all accounted for by the BRM. Better quantitative agreement would be expected with a BRM which would include a pore size distribution, corresponding to a more realistic model of a gel, but a simple uniform gel model already describes most observed effects.. A number of power laws predicted by the BRM still need to be checked experimentally, however. 

The biased reptation model provides a good framework to discuss the experimental results of the various gel electrophoresis techniques used to separate nucleic acids. Although more experiments are needed to fully characterize these techniques, available results indicate that the simplified version of the model discussed in this paper is satisfactory when low-frequency pulsed fields are used, or when transient intra-tube effects are not dominant. This is the case in continuous fields, for small molecules in intermittent fields, and possibly also for crossed fields. However, intra-tube effects are observed to play a role in field-inversion electrophoresis, for long molecules in intermittent fields, and during the first stages of an experiment (where an orientation overshoot is observed). 

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