The electric field dependence of DNA mobilities in agarose gels: A reinvestigation

Holmes, Diana L.; Stellwagen, Nancy C.

doi:10.1002/elps.1150110103

Cited by 92 publications

(43 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The pore size of the polyacrylamide gels like the ones used in this study are about one order of magnitude smaller than the ones in agarose gels (21,22). However, recent publications indicate that the pore size in polyacrylamide gels might be larger as estimated earlier (23).…”

Section: Discussionmentioning

confidence: 55%

Field inversion gel electrophoresis in denaturing polyacrylamide gels

Heller

Beck

1992

Nucl Acids Res

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 55%

Field inversion gel electrophoresis in denaturing polyacrylamide gels

Heller

Beck

1992

Nucl Acids Res

View full text Add to dashboard Cite

show abstract

“…Here, a full-length DNA molecule must occupy several gel pores, with each gel pore being large enough to accommodate multiple flexible segments of DNA, as depicted in Figure 2.1b. In order to satisfy these conditions, we use 1% agarose in 10 mM KCl, which results in an estimated dgel of ~200-400 nm [98,99], interfaced with a nanopore 3.6 nm in effective diameter, to which we introduce 5 kbp dsDNA (Rg ~470…”

Section: Nanopores Interfaced With Agarose Gelmentioning

confidence: 99%

Interfacing solid‐state nanopores with gel media to slow DNA translocations

et al. 2015

View full text Add to dashboard Cite

We demonstrate the ability to slow DNA translocations through solid‐state nanopores by interfacing the trans side of the membrane with gel media. In this work, we focus on two reptation regimes: when the DNA molecule is flexible on the length scale of a gel pore, and when the DNA behaves as persistent segments in tight gel pores. The first regime is investigated using agarose gels, which produce a very wide distribution of translocation times for 5 kbp dsDNA fragments, spanning over three orders of magnitude. The second regime is attained with polyacrylamide gels, which can maintain a tight spread and produce a shift in the distribution of the translocation times by an order of magnitude for 100 bp dsDNA fragments, if intermolecular crowding on the trans side is avoided. While previous approaches have proven successful at slowing DNA passage, they have generally been detrimental to the S/N, capture rate, or experimental simplicity. These results establish that by controlling the regime of DNA movement exiting a nanopore interfaced with a gel medium, it is possible to address the issue of rapid biomolecule translocations through nanopores—presently one of the largest hurdles facing nanopore‐based analysis—without affecting the signal quality or capture efficiency.

show abstract

“…( 1) where m 0 is the mobility in free solution (no gel), R p the average pore size, and R s is the effective coil size of the DNA molecules, which for native DNA is best described by the radius of gyration [20]. According to the Ogston model a decrease in coil size is thus expected to increase the DNA velocity exponentially, because the smaller coils will collide less frequently with gel fibers.…”

mentioning

confidence: 99%

Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromide

et al. 2005

View full text Add to dashboard Cite

Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromideWe use agarose gel electrophoresis to characterize how the monovalent catioinic surfactant cetyltrimethylammonium bromide (CTAB) compacts double-stranded DNA, which is detected as a reduction in electrophoretic DNA velocity. The velocity reaches a plateau at a ratio R = 1.8 of CTAB to DNA-phosphate charges, i.e., above the neutralization point, and the complexes retain a net negative charge at least up to R = 200. Condensation experiments on a mixture of two DNA sizes show that the complexes formed contain only one condensed DNA molecule each. These CTAB-DNA globules were further characterized by time-resolved measurements of their velocity inside the gel, which showed that CTAB does not dissociate during the migration but possibly upon entry into the gel. Using the Ogston-model for electrophoresis of spherical particles, the measured in-gel velocity of the globule is quantitatively consistent with CTAB having two opposite effects, reduction of both the electrophoretic charge and DNA coil size. In the case of CTAB the two effects nearly cancel, which can explain why opposite velocity shifts (globule faster than uncomplexed DNA) have been observed with some catioinic condensation agents. Dissociation of the complexes by addition of anionic surfactants was also studied. The DNA release from the globule was complete at a mixing ratio between anionic and cationic surfactants equal to 1, in agreement with equilibrium studies. Circular DNA retained its supercoiling, and this demonstrates a lack of DNA nicking in the compaction-release cycle which is important in DNA transfection and purification applications. IntroductionComplexation with cationic lipids is one strategy for delivery of DNA to cells. Binding of the lipids leads to compaction of the DNA coils and to a reduction of its surface charge. Both effects are believed to contribute to the facilitated uptake of the nucleic acids through the cellular membrane [1][2][3][4][5]. Methods to monitor size and charge of the complexes are therefore helpful tools in the process of screening protocols for packaging the DNA before in vivo experiments are performed. Commonly a titration series is made where the ratio R between concentration of charges from cationic lipid and DNA-phosphate groups is varied, usually in a range that includes the point R = 1 where the number of added positive and negative charges is equal. It is widely accepted that surfactants display a very strong associative binding with DNA inducing its compaction [6][7][8], aggregation [9], and precipitation [8,10,11]. The associative phase behavior presented by these systems and the absence of redissolution with the addition of an excess of surfactant lead to the assumption that the formed complexes in solution are neutral [8,12].Electrophoresis has been used to study the properties of lipoplexes because the mobility reflects their net charge. Mobilities in free solution have been used [13,14] to q...

show abstract

The electric field dependence of DNA mobilities in agarose gels: A reinvestigation

Cited by 92 publications

References 50 publications

Field inversion gel electrophoresis in denaturing polyacrylamide gels

Field inversion gel electrophoresis in denaturing polyacrylamide gels

Interfacing solid‐state nanopores with gel media to slow DNA translocations

Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromide

Contact Info

Product

Resources

About