Capillary electrophoretic separation of uncharged polymers using polyelectrolyte engines

McCormick, Laurette C.; Slater, Gary W.; Karger, Achim E.; Vreeland, Wyatt N.; Barron, Annelise E.; Desruisseaux, Claude; Drouin, Guy

doi:10.1016/s0021-9673(01)00990-6

Cited by 31 publications

(51 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since both the mobility and the a value may be strongly affected by the solution ionic strength, the free-draining model may not be entirely accurate [7,8]; however, the use of this model equation for calculating the a value enables us to at least estimate a, and also to compare directly the effective a values of our protein polymers with that reported for streptavidin, which was obtained using the same equation [16].…”

Section: Calculation Of Hydrodynamic Friction: the Ae Valuementioning

confidence: 99%

“…This approach is known as end-labeled free-solution electrophoresis (ELFSE) [4], and this perturbing entity can be called a "drag-tag" [5]. Since the time that the ELFSE concept was proposed, and the theoretical aspects of this concept were examined [4,[6][7][8], researchers have shown that this bioconjugate approach is potentially useful for the separation of short oligonucleotides [5,[9][10][11] as well as long dsDNA fragments [12]. ELFSE is a particularly promising method for DNA analysis requiring a high-resolution, size-based separation of relatively small DNA fragments, such as for genotyping or sequencing, by either capillary or microfluidic chip electrophoresis.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis

2005

View full text Add to dashboard Cite

We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.

show abstract

Section: Calculation Of Hydrodynamic Friction: the Ae Valuementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis

2005

View full text Add to dashboard Cite

show abstract

“…A theoretical model, similar to the one for ELFSE, was developed for FSCE [98], wherein for the most favourable, diffusion-limited electrophoresis conditions, an optimal DNA size for separation of the neutral polymer was predicted. The theory, being in good agreement with the available preliminary experimental results, also offers a means of estimating the persistence length of the uncharged polymer through mobility measurements.…”

Section: Electrophoresis Of Composite Moleculesmentioning

confidence: 99%

Theory of DNA electrophoresis (∼ 1999 –2002 ½)

et al. 2002

View full text Add to dashboard Cite

Over the last two decades, the introduction of new methods such as pulsed-field gel electrophoresis and capillary array electrophoresis has made it possible to map and sequence entire genomes, including our own. The development of these experimental methods has been helped by the progress of theoretical and computational sciences, and the interactions between these three modi operandi of modern science are still pushing the limits of our technologies. We now see a clear trend towards proteomics and microfluidic (even nanofluidic!) devices. In this review, we take a look at the progress of the field over the last 3 years using the glasses of the theoretical scientist and focusing mostly on new ideas and concepts. About a dozen different subfields are discussed and reviewed. We conclude by giving a commented list of some of the best review articles published over the last 2-3 years.

show abstract

“…This technique cleverly uses an uncharged "label" or "drag" molecule attached to each ssDNA chain in order to break the local balancing between friction and electric force [2-6] which normally leads to comigration of all ssDNA lengths [7,8] (excepting very small fragments [9, 10]) in free solution. More recently, a complementary technique called free-solution conjugate electrophoresis (FSCE) has been used to characterize uncharged, water-soluble polymers that can be uniquely conjugated to ssDNA [11][12][13]. Here, the ssDNA chains are of uniform length, and act as engines to pull the varying lengths of uncharged polymers for electrophoresis leading to single-monomer resolution over a wide range of molecular sizes.…”

mentioning

confidence: 99%

“…Here, the ssDNA chains are of uniform length, and act as engines to pull the varying lengths of uncharged polymers for electrophoresis leading to single-monomer resolution over a wide range of molecular sizes. In fact, the resolution obtained was approximately five times higher, and the separation efficiencies were increased by 150% compared to the more traditional RP-HPLC [12].For both FSCE and ELFSE, the theoretical equation utilized for the overall mobility m of the charged-uncharged block copolymer was until now a uniformly weighted average [5,6,11,13]:where M c is the number of charged monomers each of mobility m 0 , and M u is the number of uncharged monomers. This equation comes from a pioneer investigation of Long and co-workers [14] into the electrophoresis of polymers containing both charged and uncharged monomers.…”

mentioning

confidence: 99%

The molecular end effect and its critical impact on the behavior of charged-uncharged polymer conjugates during free-solution electrophoresis

McCormick

Slater

2005

Electrophoresis

View full text Add to dashboard Cite

Recently two novel techniques using free-solution electrophoresis to separate charged-uncharged polymer conjugates have proven successful: end-labeled free-solution electrophoresis (ELFSE) for DNA sequencing, and free-solution conjugate electrophoresis (FSCE) for molar mass profiling of uncharged polymers. The approach taken to analyze the experimental data was an extension of the theory of Long and co-workers (Long, D., Dobrynin, A. V., Rubinstein, M., Ajdari, A., J. Chem. Phys. 1998, 108, 1234-1244) for the electrophoresis of molecules with varying charge distributions. This theory also predicts that the ends of the polymers play a large role in determining the polymer's overall mobility; however, this aspect of the theory was neglected in previous work. Until now this "end effect" has, to the knowledge of the authors, not been recognized in experimental data. Through a careful investigation of the predicted end effect and a reanalysis of the experimental data, we demonstrate that indeed this effect critically impacts on the behavior of charged-uncharged polymer conjugates during electrophoresis. This work indicates that not only does the end effect need to be taken into account to avoid significant errors in data analysis, but also it provides novel system optimization approaches.

show abstract

Capillary electrophoretic separation of uncharged polymers using polyelectrolyte engines

Cited by 31 publications

References 11 publications

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis

Theory of DNA electrophoresis (∼ 1999 –2002 ½)

The molecular end effect and its critical impact on the behavior of charged-uncharged polymer conjugates during free-solution electrophoresis

Contact Info

Product

Resources

About