The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis

Yarmola, Elena G.; Sokoloff, Helen; Chrambach, Andreas

doi:10.1002/elps.1150170904

Cited by 29 publications

(22 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In other words, the band spreading departs from the Brownian diffusion. This ªenhanced diffusionº or ªdispersionº during electrophoresis was experimentally confirmed by Tinland and by Yarmola et al [6,7] for double-stranded DNA in agarose gels. A recalculation of the dispersion coefficient, based on the ªbiased reptation with fluctuationsº model (BRF), yields three regimes: (i) a plateau regime at very small electric field strength with the dispersion coefficient being equal to the field-free diffusion coeffcient (i.e.…”

Section: Introductionsupporting

confidence: 65%

Separation of double-stranded and single-stranded DNA in polymer solutions: II. Separation, peak width and resolution

Heller¹

1999

Electrophoresis

View full text Add to dashboard Cite

The electrophoretic separation of single‐stranded and double‐stranded DNA has been examined, using a matrix of linear poly‐N,N‐dimethylacrylamide (pDMA). The dependence of peak spacing, peak width and resolution on important parameters such as polymer concentration, polymer chain length and electric field strength, has been studied. This work complements our systematic study on electrophoretic mobility under different conditions (C. Heller, Electrophoresis 1999, 20, 1962—1977), and will help to further optimize and improve high performance DNA separation in capillary electrophoresis with entangled polymer solutions.

show abstract

Section: Introductionsupporting

confidence: 65%

Separation of double-stranded and single-stranded DNA in polymer solutions: II. Separation, peak width and resolution

Heller¹

1999

Electrophoresis

View full text Add to dashboard Cite

show abstract

“…However, in velocity assumed to be independent of x. The amount order to incorporate both dispersion and the observed leaving across face x + dx is [98] asymmetry of DNA peaks, a dispersion coefficient as a function of position Ddls(x) must be substituted for D so as to introduce a third moment into the shape of the migrating peak. The topic of variable coefficients in the diffusion equation is discussed by Richtmyer and (6A)…”

Section: Discussionmentioning

confidence: 99%

Theoretical studies on the mobility‐shift assay of protein‐DNA complexes

Cann

1998

Electrophoresis

View full text Add to dashboard Cite

The theory of mass transport coupled to reversible macromolecular interactions under chemical kinetic control forms the basis for computer simulation of the electrophoretic mobility-shift behavior of protein-DNA complexes. Model systems include (i) specific binding of a univalent protein molecule to a single site on the DNA molecule; (ii) the putative cage effect; (iii) cooperative binding to multiple sites; (iv) formation of looped complexes of 1:1 and 2:1 stoichiometry; (v) noncooperative and cooperative, nonspecific binding modes; and (vi) binding of dimerizing transcriptional factors to response elements of target genes. Favorable comparison of simulated with experimental mobility-shift behavior indicates that the phenomenological mechanisms, whereby observed mobility-shift patterns are generated during electrophoresis, are embodied in the theory. These studies have provided guidelines for definitive interpretation of mobility-shift assays and for the design of experiments to develop a detailed understanding of the particular system under investigation.

show abstract

“…Thus molecular crowding and collisional interactions probably are responsible for the slowed DNA diffusion. Yarmola et al (26) measured DNA diffusion in a 1% agarose gel from band spreading in the absence of an electric field. The DNA diffusion coefficient decreased from 1.7 to 0.2 ϫ 10 Ϫ8 cm 2 /s for DNA size of 1-3 kilobases.…”

Section: Discussionmentioning

confidence: 99%

Size-dependent DNA Mobility in Cytoplasm and Nucleus

Lukacs¹,

Haggie²,

Seksek³

et al. 2000

Journal of Biological Chemistry

679

478

View full text Add to dashboard Cite

The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors.The diffusional mobility of DNA fragments in cytoplasm is thought to be an important determinant of the efficacy of DNA delivery in gene therapy and antisense oligonucleotide therapy (1-3). Liposome-mediated gene transfer involves endocytic uptake, release from endosomes, dissociation of DNA from lipid, diffusion through cytoplasm, transport across nuclear pores, and diffusion to nuclear target sites (4 -7). Although considerable attention has been given to the mechanisms of cellular DNA internalization, nuclear uptake, and subsequent molecular events, little is known about the diffusive properties of introduced DNA fragments in cytoplasm and nucleus. It is not known whether the diffusion of DNA fragments is hindered by binding and steric interactions or how the size and physical structure of DNA affect its diffusional properties.Recent studies have provided information about the diffusional mobilities of small and macromolecule-sized solutes in cytoplasm and nucleus. Spot photobleaching measurements indicated that small solutes diffuse freely and rapidly in cytoplasm and nucleus, with diffusion coefficients only 3-4 times lower than that in water (8, 9). Analysis of the individual factors slowing solute diffusion, including fluid-phase viscosity, binding, and collisional interactions, indicated that the principal barrier for diffusion of small solutes was collisional interactions due to macromolecular crowding (8). The "fluid-phase" viscosity of cytoplasm and nucleus, defined as the viscosity sensed by a small probe that does not interact with cellular components, was determined by time-resolved anisotropy (10) and ratio imaging of a viscosity-sensitive fluorescent probe (11) to be only 1.2-1.4 times greater than the viscosity of water. The translational diffusion of larger, macromolecule-sized solutes (FITC 1 -labeled dextrans and Ficolls) in cytoplasm and nucleus was only 3-4-fold slower than in water for solutes Ͻ500 -750 kDa (12) but was markedly slowed for larger solutes (11, 12). The diffusional mobilities of targeted green fluorescent protein chimeras have been measured recently i...

show abstract

The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis

Cited by 29 publications

References 13 publications

Separation of double-stranded and single-stranded DNA in polymer solutions: II. Separation, peak width and resolution

Separation of double-stranded and single-stranded DNA in polymer solutions: II. Separation, peak width and resolution

Theoretical studies on the mobility‐shift assay of protein‐DNA complexes

Size-dependent DNA Mobility in Cytoplasm and Nucleus

Contact Info

Product

Resources

About