Separating DNA sequencing fragments without a sieving matrix

Ren, Hongji; Karger, Achim E.; Oaks, Frank; Menchen, Steve; Slater, Gary W.; Drouin, Guy

doi:10.1002/(sici)1522-2683(19990801)20:12<2501::aid-elps2501>3.0.co;2-h

Cited by 72 publications

(100 citation statements)

References 18 publications

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“…Perhaps the largest impact of these results is for the prediction of the optimal performance of both end-labeled free-solution electrophoresis (ELFSE) and free-solution conjugate electrophoresis (FSCE). ELFSE [31] uses a molecular drag-parachute to achieve the separation of DNA molecules in free-solution CE, while FSCE uses a DNA engine to separate a polydisperse polymer solution [32]. In both cases, the diffusion coefficient must be given by the appropriate Zimm expression, and not by the Nernst-Einstein equation.…”

Section: Discussionmentioning

confidence: 99%

Diffusion coefficient of DNA molecules during free solution electrophoresis

Nkodo¹,

Garnier²,

Tinland³

et al. 2001

Electrophoresis

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The free-draining properties of DNA normally make it impossible to separate nucleic acids by free-flow electrophoresis. However, little is known, either theoretically or experimentally, about the diffusion coefficient of DNA molecules during free-flow electrophoresis. In fact, many authors simply assume that the Nernst-Einstein relation between the mobility and the diffusion coefficient still holds under such conditions. In this paper, we present an experimental study of the diffusion coefficient of both ssDNA and dsDNA molecules during free-flow electrophoresis. Our results unequivocally show that a simplistic use of Nernst-Einstein's relation fails, and that the electric field actually has no effect on the thermal diffusion process. Finally, we compare the dependence of the diffusion coefficient upon DNA molecular size to results obtained previously by other groups and to Zimm's theory.

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Section: Discussionmentioning

confidence: 99%

Diffusion coefficient of DNA molecules during free solution electrophoresis

Nkodo¹,

Garnier²,

Tinland³

et al. 2001

Electrophoresis

Self Cite

189

158

View full text Add to dashboard Cite

show abstract

“…With the first ELSFE separations, resolution was good enough to distinguish dsDNA molecules differing by only 8 bp (for 100 bp long fragments) [4]. By improving label and coating, resolution could be pushed to 1 b size difference, enabling to sequence ssDNA up to 100 bases long [5]. So far, the limiting factors are the sample injection and the diffusion, i.e., the same factors that limit separation in polymer solutions (see below).…”

Section: Free Solution Electrophoresismentioning

confidence: 99%

Principles of DNA separation with capillary electrophoresis

2001

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“…The label was the protein streptavidin, selected because of its near zero electric charge and because it can be easily attached to DNA via the biotin complex. This novel separation principle has been adapted to DNA sequencing by Ren et al [54], who proved the possibility of sequencing up to 100 bases (in the absence of a sieving matrix!) after the primer, at fields of 333 V/cm.…”

Section: Dna Sequencingmentioning

confidence: 99%

Recent progress in DNA analysis by capillary electrophoresis

Righetti¹,

Gelfi²,

D’Acunto³

2002

Electrophoresis

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A number of recent developments in DNA analysis by capillary electrophoresis are here reviewed. They include capillary arrays for fast, parallel DNA sequencing as well as microfabricated capillary arrays. Microfluidic chips for DNA sizing and quantitation are also covered, as well as microdevices containing arrays of regular obstacles acting as size-separators during DNA migration. Screening of DNA point mutations by two much improved techniques is also reported: in one case, such mutations are detected (but only on relative short, ca. 60-70 base-long fragments) by free electrophoresis in rather acidic (pH ca. 3) buffers; in the case of single-strand chain polymorphism, an improved technique is described based on near-neutral pH buffers with mixtures of Tris/MES cations/zwitterions. When studying the behavior of inorganic and organic cations in the Debye-Hückel layer of DNA, it was found that the latter (especially a large number of Good's buffers and other zwitterions, such as His) would bind to the DNA filament not only via charge interaction, but also via additional bonds, notably hydrogen bonds, thus altering the electrophoretic (and possibly the biological) behavior of DNA molecules. However, whether or not borate ions would bind to DNA remains still an unsettled question. Finally, capillary electrophoresis was found to be instrumental in measuring fine physicochemical parameters pertaining to DNA polyelectrolytes, such as their free mobility and their translational diffusion coefficients.

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Separating DNA sequencing fragments without a sieving matrix

Cited by 72 publications

References 18 publications

Diffusion coefficient of DNA molecules during free solution electrophoresis

Diffusion coefficient of DNA molecules during free solution electrophoresis

Principles of DNA separation with capillary electrophoresis

Recent progress in DNA analysis by capillary electrophoresis

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