Theory of DNA Sequencing Using Free-Solution Electrophoresis of Protein-DNA Complexes

Mayer, Pascal; Slater, Gary W.; Drouin, Guy

doi:10.1021/ac00082a029

Cited by 87 publications

(108 citation statements)

References 18 publications

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“…In the framework of the model of ELFSE (suggested in [13]), we assume that the label (streptavidin in our case) ªcarriesº a friction coefficient equivalent to a³0 uncharged nucleotides as well as a charge equivalent to b³0 charged nucleotides. If the DNA molecule has M bases, the charge of the hybrid object increases like M+b while its friction coefficient increases like M+a instead.…”

Section: Theoretical Elementsmentioning

confidence: 99%

“…Therefore, one might expect the larger DNA molecules to elute earlier, and free-flow separation of DNAs to result. Using a simple model, Mayer, Slater and Drouin [13] were the first to predict exciting possibilities for this method, which they called end-labeled free-solution electrophoresis (ELFSE). Although many researchers found this bold proposal to be promising, the difficulties related to selecting the proper label, attaching it to the DNA, and avoiding interactions with the capillary walls, were thought to be intractable.…”

Section: Introductionmentioning

confidence: 99%

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Separating DNA sequencing fragments without a sieving matrix

et al. 1999

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Section: Theoretical Elementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Separating DNA sequencing fragments without a sieving matrix

et al. 1999

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“…Theoretical approaches to the electrophoretic separations of charged oligomers can be traced back to the modeling of their mobilities, but only few studies directly devoted to oligomers have been reported so far [14,16]. Besides, distinct electrophoretic behaviors have been recognized for small charged molecules on the one hand and for polyelectrolytes on the other.…”

Section: General Considerationsmentioning

confidence: 99%

From small charged molecules to oligomers: A semiempirical approach to the modeling of actual mobility in free solution

Cottet¹,

Gareil²

2000

Electrophoresis

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“…However, it was first suggested in the early 1990s that the constant charge-tofriction ratio of DNA that prevents its free-solution separation by electrophoresis could be overcome if DNA fragments were attached to a perturbing entity [2,3]. 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].…”

Section: Introductionmentioning

confidence: 99%

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

2005

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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.

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Theory of DNA Sequencing Using Free-Solution Electrophoresis of Protein-DNA Complexes

Cited by 87 publications

References 18 publications

Separating DNA sequencing fragments without a sieving matrix

Separating DNA sequencing fragments without a sieving matrix

From small charged molecules to oligomers: A semiempirical approach to the modeling of actual mobility in free solution

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

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