Free-solution electrophoresis of DNA

Heller, Christoph; Slater, Gary W.; Mayer, Pascal; Dovic̀hi, Norman J.; Pinto, Devanand M.; Viovy, Jean‐Louis; Drouin, Guy

doi:10.1016/s0021-9673(97)00656-0

Cited by 88 publications

(122 citation statements)

References 27 publications

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“…Therefore, a majority of the discussion was focused on large DNA separations. Figure 2 presents the separation results of dT 5 , dT 10 , dT 15 and dT 20 in gel-free solutions using a 46-cm-long (42-cm effective) and 500-nm-radius capillary. The sample was hydrodynamically injected under a pressure of 90 psi for 10 s, and the separation was executed under the same pressure for elution.…”

Section: Resultsmentioning

confidence: 99%

“…Single-standed DNA (ssDNA) were fluorescent dye (FAM) labeled deoxythymidine (dT) oligonucleotides (dT 5 , dT 10 , dT 15 and dT 20 , in which the subscripts denote the numbers of bases in the oligonucleotides) purchased from Integrated DNA Technologies (Coralville, IA). A 100 base-pair (bp) ladder was from Amersharm Biosciences (Piscataway, NJ), and a 1-kilobase pair (kbp) DNA ladder was obtained from New England Biolabs (Ipswich, MA).…”

Section: Materials and Reagentsmentioning

confidence: 99%

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Bare Nanocapillary for DNA Separation and Genotyping Analysis in Gel-Free Solutions without Application of External Electric Field

Wang

Veerappan

et al. 2008

Anal. Chem.

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In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a ~50-cm-long and 500-nm-radius bare fused silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence the operation cost is low.New, more cost-effective DNA separation methods are being sought to meet the need for simple and inexpensive assays for research and diagnostic purposes. Traditionally, DNA separations have been performed using slab-gel electrophoresis. A shift to capillary gel electrophoresis (CGE) 1 or capillary array electrophoresis (CAE) 2-4 has resulted in improved resolution and increased throughput. Both CGE and CAE use viscous polymer solutions (e.g., entangled solutions of linear polyacrylamide) as sieving matrices for size-based DNA separations. In addition to their cost, high pressures (e.g. 1000 psi) are often needed 5 to load and replenish these matrices after each run. Frequently, a coating is required on the inner wall of the capillary in order to obtain high quality separation results.To overcome the problems associated with the viscous polymer matrices, one would wish to separate DNA in gel-free (or free) solutions. 6-12 Unfortunately, DNA separations cannot normally be achieved by electrophoresis in gel-free solutions, 13 because the electrophoretic mobilities of all DNA molecules are virtually identical. Although a long DNA molecule possesses greater negative charge than a shorter molecule does, providing stronger pull, its large size induces more friction that limits its migration. These two forces largely balance one another, resulting in a mobility that is independent of the DNA size. Credit should be given to Noolandi 6 who suggested in 1992 that DNA could be electrophoretically separated in a gelfree solution if the molecules were attached to a monodisperse perturbing entity or a "dragtag". Becaus...

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Section: Resultsmentioning

confidence: 99%

Section: Materials and Reagentsmentioning

confidence: 99%

Bare Nanocapillary for DNA Separation and Genotyping Analysis in Gel-Free Solutions without Application of External Electric Field

Wang

Veerappan

et al. 2008

Anal. Chem.

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“…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. The ELFSE concept was demonstrated to be valid in 1998 when the separation of 100-base dsDNA ladder (up to & 1000 bases) was achieved in free-solution CE [14]. The label was the protein streptavidin (selected because of its near-zero electric charge and the fact that it can easily be attached to DNA via the biotin complex).…”

Section: Introductionmentioning

confidence: 99%

Separating DNA sequencing fragments without a sieving matrix

et al. 1999

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“…This ªresolution lengthº is equivalent to the ªseparation factorº defined in [14], but reciprocal to the ªresolution per base pairº defined in [15]. Sutherland et al [16] have introduced the ªresolving powerº, R, which is equal to the DNA size divided by RSL.…”

Section: Introductionmentioning

confidence: 99%

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

Heller¹

1999

Electrophoresis

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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.

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Free-solution electrophoresis of DNA

Cited by 88 publications

References 27 publications

Bare Nanocapillary for DNA Separation and Genotyping Analysis in Gel-Free Solutions without Application of External Electric Field

Bare Nanocapillary for DNA Separation and Genotyping Analysis in Gel-Free Solutions without Application of External Electric Field

Separating DNA sequencing fragments without a sieving matrix

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

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