Transverse Pore Gradient Gel Research Articles

Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.

Pulsed field electrophoresis for the separation of protein-sodium dodecyl sulfate-complexes in polyacrylamide gels.

Polyacrylamide gel electrophoresis of proteins was studied using a pulsed-current mode. A new "local field" distribution was used to correct the gel patterns and optimize migration. A corrective field was applied at fixed 2 s intervals to a constant field, inducing a complex relaxation mechanism. Calculated variations in the local field directions decreased the electric strain on the gel during the run, with resultant optimum gel structure. The relaxation mechanism was found to enhance the absolute mobility of proteins with shorter running times compared to constant field gel electrophoresis (CFGE) and other pulsed field techniques. The enhancement of molecular mobility was explored by transverse pore gradient gel electrophoresis. Ferguson curves which exhibited a convex shape in CFGE were linearized by the new pulsed-field method named pulsed oscillatory high-performance electrophoresis (POPE).

Transverse pore gradient gel electrophoresis, using the PhastSystem.

The application of pore gradient gels prefabricated for the PhastSystem (Pharmacia) to transverse pore gradient gel electrophoresis is demonstrated. It has the twofold advantage of (i) horizontal positioning, avoiding gel stretching during the preparation of these gels and resulting pore size irreproducibility experienced with vertically applied pore gradient gels, which necessitate an orthogonal transfer of spacers, and (ii) miniaturized gel dimensions, which allow a small sample load and a short duration of electrophoresis and staining.

A three-dimensional plot for the display of transverse pore gradient gel electrophoresis data: application to a kinetoplast DNA fragment of planar circular conformation.

A 219 bp fragment of kinetoplast DNA from Crithidia fasciculata has previously been shown to exhibit a gel concentration dependent, anomalously low migration rate in 3-10% polyacrylamide transverse pore gradient gels relative to that of straight standards. The anomaly was interpreted in accordance with previous results of Marini et al. [1] as being due to the planar circular conformation of the fragment. The transverse pore gradient gel pattern is displayed here as a three-dimensional plot of migration distance vs. gel concentration and DNA length for the kinetoplast fragment and straight DNA standards. The plot yields a smooth surface for the standards clearly separated from the curve of the kinetoplast DNA fragment. Thus, transverse gradient gel electrophoresis in conjunction with the three-dimensional plot provides a unique method for the recognition of abnormal conformational features of DNA on the basis of electrophoretic mobility in a pore-size gradient.

How far have we progressed toward automated electrophoresis in sieving media of the twenty-first century?

The automation of electrophoresis in polymeric sieving media requires (i) an objective definition of the conditions (the polymer, its concentration, solvent, buffer, pH, ionic strength, temperature) under which a particular separation proceeds most effectively; (ii) apparatus capable of zone detection, acquisition by computer, evaluation (migration distance, zone width and area) and a print-out of the number of components, their size and net charge, and the polymer conditions under which each component separates most effectively from its two neighboring zones. Both of these prerequisites of automation have been met to a first approximation at this time and, after further maturation, assembly and streamlining should be able to fill the need of the coming century for a more efficient, nonarbitrary and cost-effective mode of macromolecular and cellular particle separation. (i) The realization of the qualitative equivalence of polymer solutions and gels has greatly increased our options in the choice of sieving media. That choice can be made objectively by correlating separation efficiency (S), particle size (R) and intrinsic viscosity (eta o) of the polymer. (S) is a function of the slope, KR(R), of the Ferguson plot [log(mobility) vs. polymer concentration], or with nonlinear plots (DNA, agarose) KR(R,T). KR is at present most easily derived from transverse pore gradient gels or by conducting capillary zone electrophoresis (CZE) at multiple polymer concentrations. Pore gradient CZE appears promising. CZE also defines the free mobility unequivocally. Computer programs exist to generate KR from migration distances (times), and optimal S and polymer concentration for a particular R from KR.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the electrophoretic properties of nucleosome core particles by transverse polyacrylamide pore gradient gel electrophoresis.

Transverse pore gradient gel electrophoresis, previously applied to bent DNA, has extended the usefulness of the gel retardation assay in two ways: (i) by differentiating between different DNA conformations; (ii) by providing information regarding the physical properties of DNA. In the present study, similarly extended information is obtained with regard to a well-characterized DNA-protein complex, the chicken erythrocyte nucleosome core particle. (i) The winding of DNA around the protein core constrains the DNA which renders its Ferguson curve (migration distance vs. gel concentration) similar to that of kinetoplast DNA, i.e. it intersects sharply with the Ferguson curves of linear DNA standards. By contrast, the deproteinized nucleosome DNA exhibits a Ferguson curve similar to linear standards of the same length. (ii) Interpretation of the Ferguson curve based on a mathematical model shows that the nucleosome exhibits a linear Ferguson plot [log(mobility) vs. gel concentration]. This is similar to and characteristic of spherical proteins, contrasting with the concave plot typical for linear and bent DNA. (iii) The effective size of the nucleosome, evaluated in terms of an "equivalent sphere" (i.e. a hypothetical spherical particle with a radius, Res, having the same electrophoretic mobility as DNA for a particular set of experimental conditions), remains invariant across the gel concentration range of 3-9%T. This is similar to proteins and bacteriophages and contrasts with the progressive decline of Res with increasing gel concentration observed for linear DNA and the deproteinized nucleosomal DNA.

Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media.

Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to "linear" standards. The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards. Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E. coli operon promoter shows in addition that curves of migration distance vs. gel concentration ("Ferguson curves") of such abnormally conformed DNA differ from those of "linear" standards. Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size. In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than "linear" standards from one which shows the same dependence. The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards. Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel. Another authentically "abnormal" DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid. Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer-aided analysis of DNA curves on transverse gradient gels

Transverse pore gradient polyacrylamide gel electrophoresisof DNA restriction fragments was used to generate gel patterns describing migration distance as a function of gel concentration (Ferguson curves). These Ferguson curves were digitized, traced and analyzed with the aid of a personal computer. The traced curves were plotted semi-logarithmically and the plots were subjected to least-squares linear regression analysis to yield values of the slope (KR) and the intercept at %T = 0 (YO). These values are highly precise since they are based on approx. 100 measurements per curve. The computerized method reduces the errors due to manual measurements of migration distances and is time and labor saving. The method is still limited to intra-experimental comparison of Ferguson curves, since it does not as yet comprise a determination of gel concentration. At present, curve tracing remains semi-automated, requiring manual intervention when Ferguson curves cross or approach one another. Potentially, the importance of the computerized analysis of transverse pore gradient gels lies in the rapid quantitative interpretation of Ferguson curves for detection of anomalously migrating DNA species. Potentially, that application provides a more sensitive and informative mode of detection than

Transverse pore gradient gel electrophoresis of lipoprotein [a] using methylenebisacrylamide gradients.

We describe a modification of transverse pore gradient gel electrophoresis in which pore size is regulated by crosslinker proportion (%C) rather than by total monomer concentration (%T). We electrophoresed plasma lipoprotein [a] transversely across linear N,N'-methylenebisacrylamide gradients and measured mobility as a function of %C. This method allows for the simultaneous assessment of pore sizes generated over a wide range of crosslinker proportions.