Rapid separation and laser‐induced fluorescence detection of mutated DNA by capillary electrophoresis in a self‐coating, low‐viscosity polymer matrix

Du, Mingrui; Flanagan, James H.; Lin, Baiquan; Ma, Yinfa

doi:10.1002/elps.200305572

Cited by 18 publications

(13 citation statements)

References 47 publications

(39 reference statements)

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“…This is in accordance with the results obtained by use of hydroxypropylmethylcellulose matrices at pH 7.5, 8.0, and 8.5, in analysis of DNA singlestrand fragment length [4]. Nevertheless, the lifetime of the capillaries deteriorates at higher pH; thus, using dynamic coating sieving matrices with pH below 8, a fivefold increase in capillary lifetime can be observed [4,5]. This is in accordance with our experiences when using commercial polymer POP-4 ™ (Applied Biosystems, Foster City, CA, USA) (data not shown).…”

Section: Resultssupporting

confidence: 92%

“…In this study we used pH-sensitive 6-FAM fluorophores, but did not notice any difference in signal height for the pH tested (data not shown). Based on previous publications, PDMA and PVP or in a combination of the two have been used to determine the size of DNA, either double or single strand [4,5,21], but not used for CTCE analysis. Thus, we formulated sieving matrices with a combination of the two polymers as shown in Table 2.…”

Section: Resultsmentioning

confidence: 99%

“…Two approaches are used to suppress EOF, either by covalently linking chains of the polymer to the silanol groups of the silica [1] or by disguising the charges with substances like those previously reviewed [2,3]. Several studies have addressed EOF suppression in DNA sequencing, fragments length analyses, thermal gradient capillary electrophoresis (TGCE), and SSCP [4][5][6][7]. However, no systematic evaluation of dynamic coating polymers used for denaturing CE (DCE) analysis of DNA variants can be found in the literature.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

Ekstrøm

Bjørheim

2006

Electrophoresis

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Denaturing CE (DCE) is a powerful tool for analysis of DNA variation. The development of commercial multi-CE instruments allows large-scale studies of DNA variation (many samples and many fragments). However, the cost of consumables like capillary arrays and sieving matrix might limit the use of DCE in such studies. Thus, we have tested 72 different in-house formulated sieving matrices' ability to suppress EOF and separate PCR-amplified alleles with the DCE variant, cycling temperature CE (CTCE). The data herein demonstrate that alleles can be baseline-separated by use of PVP and poly(N,N-dimethyl acrylamide) polymers at various percentages and pH. Allele separation by CTCE is matrix-independent and consequently applicable to any capillary instrument used for DNA separation. Formulation of sieving matrix for CTCE was done by dissolving appropriate amount of polymer powder into the running buffers. Allele separation was observed at different pH (7.5-8.5), concentrations and molecular size of the polymer, without compromising the separation and reproducibility. Finally, the cost reduction of homemade matrices is more than 1000-fold as compared to commercial sieving matrices.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

Ekstrøm

Bjørheim

2006

Electrophoresis

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“…[10][11][12][13][14]. These devices are frequently employed for the separation of samples using a technique which is similar to that used in conventional capillary electrophoresis analysis, but which is more efficient and more rapid [15,16].…”

Section: Introductionmentioning

confidence: 99%

Numerical simulation of electrokinetic injection techniques in capillary electrophoresis microchips

et al. 2005

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The effective design and control of a capillary electrophoresis (CE) microchip requires a thorough understanding of the electrokinetic transport phenomena associated with its microfluidic injection system. The present study utilizes a numerical simulation approach to investigate these electrokinetic transport processes and to study the control parameters of the injection process. Injection systems with a variety of different configurations are designed and tested, including the cross-form, T-form, double-T-form, variable-volume focused flow cross-form, and variable-volume triple-T-form configuration. Each injection system cycles through a predetermined series of steps in which the magnitudes and distributions of the applied electric field are precisely manipulated in order to effectuate a virtual valve. This study investigates the sample leakage effect associated with each of the injection configurations and applies the double-L, pullback, and focusing injection techniques to minimize the sample leakage effect. The injection methods presented in this paper have the exciting potential for use in high-quality, high-throughput chemical analysis applications and throughout the micro-total-analysis systems field.

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“…[20][21][22][23][24] We tested three different capillaries in this study. One of them was recovered with PVA, a highly hydrophilic polymer covalently bound to the capillary surface.…”

Section: Capillary Inner Coatingsmentioning

confidence: 99%

Evaluation of capillaries with different inner coatings for DNA analysis using dilute polymer solutions by capillary electrophoresis

Eugênio¹,

Carrilho²

2009

J. Braz. Chem. Soc.

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No presente trabalho, três colunas capilares, uma sem recobrimento interno e duas com recobrimentos internos diferentes foram utilizadas na separação de fragmentos de DNA: poli(vinil álcool) (PVA) e poli(dimetilacrilamida) (PDMA) -ambos de recobrimento covalente -foram comparados para a separação de DNA utilizando soluções poliméricas. As separações foram realizadas usando hidroxietilcelulose (HEC) (90-105 kDa) nas concentrações entre 0,00 e 2,00% m/v. Os resultados indicaram que a eficiência de separação foi maior no capilar de PVA do que no de PDMA, em todas as concentrações de HEC testadas. Ainda, uma resolução superior também foi observada com o capilar de PVA, já que com o capilar de PDMA o formato dos picos não se mostrou reprodutível quando corridas subseqüentes foram realizadas. Contrariamente ao relatado na literatura, nenhuma separação foi conseguida com o capilar sem revestimento interno.In this work three capillary columns, one with uncoated inner wall and two with covalentlybound internal coatings -poly(vinyl alcohol) (PVA) and poly(dimethylacrylamide) (PDMA) -both covalently covered -were used to separate DNA fragments and compared to DNA separation using replaceable polymer solutions. The separations were performed using hydroxyethylcellulose (HEC) (90-105 kDa) in concentrations ranging from 0.00 to 2.00% m/v. The results indicated that the separation efficiency was higher in the PVA capillary than in the PDMA in all evaluated concentrations of HEC. In addition, higher resolution was also observed in PVA-coated capillary since in PDMA the shape of the peaks was not reproducible when subsequent runs were performed. Contrary to what has previously been reported in the literature, no reasonable separation was possible in bare fused silica.Keywords: DNA separation, capillary coating, laser-induced fluorescence, PDMA, PVA IntroductionCapillary electrophoresis (CE) is a separation technique widely used for separation of biological macromolecules. It offers great advantages over slab-gel electrophoresis (SGE) -shorter analysis time, smaller quantity of sample, greater efficiency of separation, and easier automation. 1,2 The first attempts to apply CE to DNA separations were carried out in capillaries filled with cross-linked gels, like polyacrylamide. [3][4][5] Later on, some polymeric solutions were used, known as dynamic or physical gels, which are easily prepared and introduced into the capillary, thus enabling its renewal after each analysis and the use of a single capillary for many separations, without any problem with the subsequent separations. 6 DNA migration mechanisms in polymeric matricesThe Ogston and the reptation models were first proposed to explain DNA migration in slab gels, but they have also shown themselves suitable for polymeric solutions. 7 According to the Ogston theory, the DNA fragment keeps a spherical and rigid conformation of radius Rg. This particle moves across a random net formed by the gel fibers. These fibers form pores with average size ξ, which is a function of the po...

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Rapid separation and laser‐induced fluorescence detection of mutated DNA by capillary electrophoresis in a self‐coating, low‐viscosity polymer matrix

Cited by 18 publications

References 47 publications

Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

Numerical simulation of electrokinetic injection techniques in capillary electrophoresis microchips

Evaluation of capillaries with different inner coatings for DNA analysis using dilute polymer solutions by capillary electrophoresis

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