End‐labeled free‐solution electrophoresis of DNA

Meagher, Robert J.; Won, Jong‐In; McCormick, Laurette C.; Nedelcu, S.; Bertrand, Martin; Bertram, Jordan L.; Drouin, Guy; Barron, Annelise E.; Slater, Gary W.

doi:10.1002/elps.200410219

Cited by 107 publications

(161 citation statements)

References 42 publications

(88 reference statements)

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“…One exciting approach that has received considerable attention is endlabeled free-solution electrophoresis (ELFSE) [16][17][18][19][20]. In this approach, DNA is modified end-on with an uncharged, monodisperse, polymeric end-label, or "dragtag" to create a charged-uncharged polymer conjugate.…”

Section: Generalmentioning

confidence: 99%

Free‐solution electrophoresis of DNA modified with drag‐tags at both ends

et al. 2006

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In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al.., J. Chromatogr. A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26, 1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.

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Section: Generalmentioning

confidence: 99%

Free‐solution electrophoresis of DNA modified with drag‐tags at both ends

et al. 2006

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“…This view is part of a larger tendency of moving away from previous technologies. For example, in DNA analysis, the chemical gel from gel electrophoresis was replaced by physical gel (polymer solution), then by dilute polymer solution, and more recently by simple BGE [18].…”

Section: Introductionmentioning

confidence: 99%

Ion-interaction–capillary zone electrophoresis of cationic proteomic peptide standards

Popa¹,

Mant

Hodges

2006

Journal of Chromatography A

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We have employed a novel capillary electrophoresis (CE) approach recently developed in our laboratory, termed ion-interaction-capillary zone electrophoresis (II-CZE), to the resolution of a mixture of 27 synthetic cationic proteomic peptide standards. These peptides were comprised of three groups of nine peptides (with net charges of +1, +2 and +3 for all nine peptides within a group), the hydrophobicity of the nine peptides within a group varying only subtly between adjacent peptides. This bidimensional CE approach achieved excellent resolution of the peptides with high peak capacity by combining the powerful CZE mechanism located in the background electrolyte (BGE) with an hydrophobicity-based mechanism also located in the BGE, the latter consisting of high concentrations (up to 0.4 M) of aqueous perfluorinated acids ( trifluoroacetic acid, pentafluoropropionic acid and heptafluorobutyric acid). Thus, concomitant with a CZE separation of the three differently charged groups of peptides, there is an hydrophobically-mediated separation of the peptides within these groups effected through interaction of the hydrophobic anions of the perfluorinated acids with hydrophobic amino acid side-chains in the peptides. This methodology is dramatically different from other CE methods that have used complexing agents such as micelles or cyclodextrins in MEKC. Overall, the results presented here demonstrate the value of CE as a peptide separative tool in its own right, including its use for proteomic applications, and not merely as a complementary technique to reversed-phase high-performance liquid chromatography (RP-HPLC).

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“…Beyond the Debye length, the counter-ions can move opposite to the polymer movement, and this sets up a hydrodynamic shear boundary and viscous dissipation. Unfortunately, under conditions where dsDNA is stable (10 mM salt), the Debye length is much smaller than the radius of gyration of a long dsDNA molecule and the polymer is said to be 'free-draining': the electrophoretic mobility is independent of the length of the dsDNA [9], which means that you cannot separate long DNA molecules as a function of length in free solution. This is why gels were developed, to bring in higher order length-dependent drag coefficients, and why we built the post array.…”

Section: Charged Polymers In Saline Solution Do Not Show a Net Chargementioning

confidence: 99%

Ratchets in hydrodynamic flow: more than waterwheels

et al. 2014

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The transport of objects in microfluidic arrays of obstacles is a surprisingly rich area of physics and statistical mechanics. Tom Duke's mastery of these areas had a major impact in the development of biotechnology which uses these ideas at an increasing scale. We first review how biological objects are transported in fluids at low Reynolds numbers, including a discussion of electrophoresis, then concentrate on the separation of objects in asymmetric arrays, sometimes called Brownian ratchets when diffusional symmetry is broken by the structures. We move beyond this to what are called deterministic arrays where non-hydrodynamic forces in asymmetric arrays allow for extraordinary separation, and we look to the future of using these unusual arrays at the nanoscale and at the hundreds of micrometre scale. The emphasis is on how the original ideas of Tom Duke drove this work forward.

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End‐labeled free‐solution electrophoresis of DNA

Cited by 107 publications

References 42 publications

Free‐solution electrophoresis of DNA modified with drag‐tags at both ends

Free‐solution electrophoresis of DNA modified with drag‐tags at both ends

Ion-interaction–capillary zone electrophoresis of cationic proteomic peptide standards

Ratchets in hydrodynamic flow: more than waterwheels

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