The upper limit of the size range of DNA amenable to separation in agarose solutions above their gelling temperature, using capillary zone electrophoresis apparatus, was increased to 12 kb. The plot of log(bp) vs. mobility derived from electrophoresis in 1.7% agarose solution is biphasic, exhibiting higher resolving power for DNA less than 1 kb in size than that of larger sizes. Resolving power for DNA larger than 1 kb increased when the agarose concentration was increased in the range of 1.0-2.6%. It was similar in solutions at 40 degrees C of SeaPrep and SeaPlaque agaroses as well as in Acrylaide (trade names are those of the manufacturer). However, the resolving power of SeaPrep agarose at 25 degrees C was inferior to that at 40 degrees C. Concave plots of log(mobility) vs. concentration of the agarose solutions are those predicted under the assumption that the effective "equivalent radius" of the DNA molecule diminishes with increasing agarose concentration in the investigated concentration range up to 2.6%.

Base Composition↗

PubMedDate not supplied

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)

Animals↗

PubMedDate not supplied

Molecular sieving of lambda phage DNA in polyacrylamide solutions as a function of the molecular weight of the polymer.

Electrophoresis of lambda phage DNA was carried out in solutions at various concentrations of uncrosslinked polyacrylamide of 0.6, 1, 5 and 9 x 10(6) molecular weight (Mw) with narrow Mw distribution. By inspection of mobilities in the various concentration ranges, it appears that mobilities decrease, and retardation increases, with increasing Mw. The relation between electrophoretic retardation and the Mw of the polymer was also interpreted (i) in the manner previously applied to nonlinear Ferguson plots and compatible with the Ogston model; and (ii) empirically, on the basis of the first derivatives of the functions describing the Ferguson plots at the polymer concentrations used. Interpretation (i) shows that the retardation increases linearly in the order of 0.6, 1, 5 and 9 x 10(6) Mw of polyacrylamide. Interpretation (ii) shows a nonlinear increase of retardation in the Mw range 5 to 9 x 10(6), and a decrease in retardation as Mw is raised from 0.6 to 5.0 x 10(6). Hypothetically, interpretation (ii) can be explained mechanistically by a progressive change, as the polymer size is increased, from a collision with the surface of the polymer fiber to one occurring after permeation in the interior of a random-coiled fiber. Interpretation (i) may fail to detect that change due to the large difference between DNA mobility in solutions of the smallest polymer and the free mobility. DNA peak detection in all of the four size classes of polyacrylamide in solution is limited to relatively narrow ranges of polymer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylic Resins↗

PubMedDate not supplied

Advances in DNA electrophoresis in polymer solutions.

DNA electrophoresis in gels and solutions of agarose and polyacrylamide was objectively evaluated with regard to separation efficiency at optimal polymer concentrations. In application to DNA fragments, polyacrylamide gels were superior for separating fragments of less than 7800 bp, and agarose gels are the best choice for larger fragments. Agarose solutions are nearly as good as polyacrylamide gels for small DNA (< 300 bp). Agarose solutions have a higher efficiency than polyacrylamide solutions for DNA of less than 1200 bp. Separation efficiency sharply decreases with increasing length of DNA. Retardation in polyacrylamide solutions was found to depend on polymer length in a biphasic fashion. The choice of resolving polymer concentrations depends on the progressive stretching of DNA in proportion to polymer concentration. The rate of that stretching appears higher in polyacrylmide solution than in gels or in liquid or gelled agarose. Application of polymer solutions to capillary electrophoresis raises further problems concerning agarose plugs, DNA interactions with the polymers, operation at low field strength and long durations as well as detection sensitivity.

Acrylic Resins↗

PubMedDate not supplied

Relative efficiency of molecular sieving in solutions of four polymers.

The efficiency of size separations of polystyrene sulfate, 120-1085 nm radius, by molecular sieving in polymer solutions, expressed by the separation efficiency function S = M0 magnitude of dKR(R)/dR Te-KR(R)T [where M0 is the mobility in free solution, KR the retardation coefficient, R the geometric mean radius of the particle, KR(R) the retardation coefficient as a function of R, and the polymer concentration is T] increases from methylhydroxypropyl cellulose to polyvinyl alcohol to uncrosslinked polyacrylamide to agarose above its gelling temperature. Separations of DNA, in the size range of 3-21 nm radius, are by at least one order of magnitude more efficient than those of polystyrene sulfate in the size range of 120-1085 nm radius. A plot of S vs. R in the experimental range of T is constructed for the four polymer solutions; this allows one to select optimal media and concentrations for the sieving of particles in the desired range of molecular sizes.

Acrylic Resins↗

PubMedDate not supplied

Capillary zone electrophoresis of large DNA.

Capillary zone electrophoresis (CZE) of DNA 23.1 to 48.5 kb in length in polyacrylamide solutions of several concentrations provides evidence for polymer concentration and DNA length-dependent stretching and orientation of these species and suggests an effective separation at a polymer concentration of about 0.6%. Applying a 0.1% polyacrylamide concentration to the lambda-phage DNA ladder, at least 5 components are separated; separation improves with lowering of the field strength to 2 V/cm and, correspondingly, extended duration of CZE. Saccharomyces pombe chromosomal DNA separates into 3 major components on CZE at high field strength (270 V/cm) in 0.9% polyacrylamide solution, confirming a previous finding made on electrophoresis in a 1.1 mm ID tube at low field strength. However, the finding is limited to one source of the DNA plug, and the chromosomal identity of the components remains unknown. Methodological problems in the CZE of large DNA relate to the need for extended duration of pressure injection if absorbance detection is applied, the need to define the starting zone after extended pressure injection, the need to melt and digest agarose plugs prior to loading, and related needs for thermostating of the sample chamber and for software compatible with low voltage operation.

Acrylic Resins↗

PubMedDate not supplied

Capabilities and potentialities of transverse pore gradient gel electrophoresis.

Transverse pore gradient gel electrophoresis is important as a tool for obtaining nonlinear Ferguson plots [log(mobility) vs. gel concentration], e.g. in application to DNA in polyacrylamide gels or to agarose gels, with the purpose of evaluating molecular properties (size, conformation, malleability) and gel fiber properties (fiber radius and length per unit volume). To date, it is capable of (i) yielding gel patterns ("Ferguson curves") of migration distance vs. predicted % T-range of the pore gradient, assuming its linearity; (ii) yielding information regarding molecular conformation from the intersection of Ferguson curves of unknowns (e.g. bent DNA) with those of standards; (iii) acquisition of Ferguson curves by computer, using prototype instrumentation; (iv) mathematical manipulation of acquired Ferguson curves to yielding Ferguson plots, providing that mobility in free solution has been assessed by capillary zone electrophoresis. The potentialities of the method remain unfulfilled to date due to (i) the unavailability, with a single exception, of an accurate and precise way to produce pore gradients of known shape; (ii) unavailability of a routinely applicable analysis for % T; (iii) unavailability of optimized, user-friendly and foolproof instrumentation for computer acquisition of Ferguson curves, including the present inapplicability of a commercially available electrophoresis apparatus with intermittent optical detection to transverse pore gradient gels; and (iv) unresolved problems in the statistical evaluation of Ferguson curves.

DNA↗

PubMedDate not supplied

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.

Base Composition↗

PubMedDate not supplied

Detection of a single base mismatch in double-stranded DNA by electrophoresis on uncrosslinked polyacrylamide gel.

Uncrosslinked polyacrylamide forms gels in the concentration range of 15-40% acrylamide. Electrophoresis in these gels of a commercially available 350 bp heteroduplex DNA preparation separates it from the homoduplex DNA of the same size. The separation is qualitatively equivalent to that previously achieved in a commercial proprietary gel ("Mutation Detection Gel" of AT-Biochem), or in an equivalent 14% T, 0.15% C (N,N'-methylenebisacrylamide) gel, but the mechanical stability of mutation detection electrophoresis (MDE) gels or 0.15% C gels is better than that of uncrosslinked polyacrylamide gels. The separation in any of these three gel media can be carried out in short gel tubes within a few hours of electrophoresis time. In both uncrosslinked polyacrylamide and MDE gel media, the Ferguson plots [log(mobility) vs. gel concentration] and the plots of effective molecular radius vs. gel concentration ("T-plots") of both the heteroduplex and homoduplex DNA indicate an augmented size but similar flexibility upon passage through the gel than exhibited by the components of a DNA standard ladder. Homoduplex and heteroduplex DNA correspondingly exhibit a parallelism of their Ferguson curves in transverse MDE pore gradient gel electrophoresis, suggesting a surface net charge difference, possibly due to a conformational reorientation too subtle to be detected by a shift in the slope of the Ferguson plot, as has been observed once previously with a "kinked" DNA species. The gel fiber radius or length per unit volume of uncrosslinked polyacrylamide and MDE gels do not differ significantly within confidence limits, which are wide compared to unconventionally crosslinked gels, presumably because of their greater swelling.

Acrylic Resins↗

PubMedDate not supplied

The relative separation efficiencies of highly concentrated, uncrosslinked or low-crosslinked polyacrylamide gels compared to conventional gels of moderate concentration and crosslinking.

The joint report [1] has shown that the separation of heteroduplex DNA from homoduplex DNA can be achieved by uncrosslinked polyacrylamide gels or gels of a very low degree of crosslinking (0.15%) with N,N'-methylenebisacrylamide (Bis), while conventional polyacrylamide gels of 2-5% crosslinking with Bis are incapable of such a separation in the absence of added denaturing agents. This result raised the question whether in application to other separation problems the same superiority of uncrosslinked or low-crosslinked polyacrylamide existed. To test that question, Ferguson plots were determined for the members of a DNA ladder (50 to 1000 bp) in polyacrylamide with 0, 0.1, 0.2, 0.3, 0.5% C (Bis), and the separation efficiency function, S, was evaluated in comparison with that in conventional 2-5% C (Bis) gels. S was found to be lower, not higher, in gels of low crosslinking at the respective maximally effective gel concentrations. However, the range of gel concentrations in which gels of low or no crosslinking were effective extended over a range of at least 10% T, while conventionally crosslinked gels were most effective over a range of 3 to 1 units of %T.

Acrylic Resins↗