Beyond Gel Electrophoresis: Microfluidic Separations, Fluorescence Burst Analysis, and DNA Stretching

Dorfman, Kevin D.; King, Scott; Olson, Daniel W.; Thomas, Joel D. P.; Tree, Douglas R.

doi:10.1021/cr3002142

Cited by 176 publications

(158 citation statements)

References 750 publications

(2,578 reference statements)

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“…Exploiting biased reptation for 2D DNA fractionation B Gumuscu et al principle states that the mobility, μ, of a DNA fragment in an agarose sieving matrix is a function of the electric field and DNA contour length 23,[25][26][27][28] . This behavior was experimentally confirmed in the μGEL device.…”

Section: Resultsmentioning

confidence: 99%

“…Periodic application of E 1 and E 2 results in the ionic species following a path at an angle Φ 0 4 Φ (Supplementary Video S5) 27 . When the applied field strengths were 59.5 V cm − 1 (E 1 ) and 22.4 V cm − 1 (E 2 ), we calculated Φ 0 = 45°for the fluorescein sodium salt, predicting clear separation from the DNA fragments with a calculated Φ o 38.5°(Supplementary Figure S2).…”

Section: Resultsmentioning

confidence: 99%

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Exploiting biased reptation for continuous flow preparative DNA fractionation in a versatile microfluidic platform

et al. 2017

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A new approach is presented for preparative, continuous flow fractionation of sub-10-kbp DNA fragments, which exploits the variation in the field-dependent mobility of the DNA molecules based on their length. Orthogonally pulsed electric fields of significantly different magnitudes are applied to a microchip filled with a sieving matrix of 1.2% agarose gel. Using this method, we demonstrate a high-resolution separation of 0.5, 1, 2, 5, and 10 kbp DNA fragments within 2 min. During the separation, DNA fragments are also purified from other ionic species. Preparative fractionation of sub-10-kbp DNA molecules plays an important role in second-generation sequencing. The presented device performs rapid high-resolution fractionation and it can be reliably manufactured with simple microfabrication procedures. This method has the advantages of simplicity, versatility, and reproducibility. However, it suffers from long processing times on the order of a few tens of hours 2,3 . For instance, the Bio-Rad CHEF-DR device performs fractionation of 5-120 kbp DNA using pulsed-field gel electrophoresis (PFGE) in 25 h (Refs. 4,5). Trends toward second-generation sequencing motivate the replacement of standard gel electrophoresis with microchip-based systems, which could provide efficient platforms to minimize the processing time and to perform optimal DNA fractionation 6-8 .Micro-and nanofabricated post arrays-that resemble a gel matrix with well-ordered and identical pores-have been integrated in microchip-based systems, increasing both the understanding of DNA separation principles and the fractionation's efficiency and speed. Two decades ago, Volkmuth and Austin 9 introduced a patterned micro-post array in a microfluidic electrophoresis platform, in which DNA fragments were separated by biased reptation under a DC electric field. Later, Duke et al. separated large fragments in a range of 60-135 kbp in a microfabricated array using DNA reorientation-or the 'switchback' principle. Although the switchback principle is similar to what is used in PFGE, the device yields much faster separations owing to its sparse and regular sieving array 10 . In a later work, Kaji et al. studied a three-dimensional (3D) nanopost array as an optimal separation matrix for DNA fragments over a few kbps under a DC electric field, resulting in similarly fast separations due to the sparse matrix 11 .With the aim of increasing the sample throughput and further facilitating the sample recovery, a continuous flow separation was developed. Huang et al. 12 performed continuous flow PFGE in a micromachined post array by applying pulsed electric fields of slightly unequal strengths. DNA fragments ranging between 61 and 209 kbp were separated within 15 s in the 'DNA prism'.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Exploiting biased reptation for continuous flow preparative DNA fractionation in a versatile microfluidic platform

et al. 2017

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“…According to the biased reputation mechanism, the electrophoretic mobility of a DNA molecule not only relies on its molecular size, but also relies on the applied electric field: (2) where N is the DNA molecular size and b is a constant. 27 The Eq. (2) clearly describes that the high applied electric field can increase the electrophoretic mobility of given DNA molecules remarkably.…”

Section: Resultsmentioning

confidence: 99%

Fast High-Throughput Screening of H1N1 Virus by Parallel Detection with Multichannel Microchip Electrophoresis

Zhang

Lee

et al. 2015

Methods in Molecular Biology

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A multi-channel microchip electrophoresis (MCME) method with parallel laser-induced fluorescence (LIF) detection was developed for rapid screening of H1N1 virus. The hemagglutinin (HA) and nucleocapsid protein (NP) gene of H1N1 virus were amplified using polymerase chain reaction (PCR). The amplified PCR products of the H1N1 virus DNA (HA, 116 bp and NP, 195 bp) were simultaneously detected within 25 s in three parallel channels using an expanded laser beam and a charge-coupled device camera. The parallel separations were demonstrated using a sieving gel matrix of 0.3% poly(ethylene oxide) (M r = 8,000,000) in 1× TBE buffer (pH 8.4) with a programmed step electric field strength (PSEFS). The method was ~20 times faster than conventional slab gel electrophoresis, without any loss of resolving power or reproducibility. The proposed MCME/PSEFS assay technique provides a simple and accurate method for fast high-throughput screening of infectious virus DNA molecules under 400 bp.

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“…In the field of electrophoresis in gels and micro and nanofluidic devices, the entropic trapping regime refers to the case where the nominal pore size in the device is commensurate with the radius of gyration of the DNA [1,12] and so PEs in their migration, periodically encounter entropic barriers. So far several designs for microfabricated channels have been introduced and used in experiments.…”

Section: Entropic Trapmentioning

confidence: 99%

“…Migration of charged polymer chains under the action of electric field, occurs in many in vivo and in vitro phenomena and experiments such as RNA migration from nucleus' pores, electrophoresis techniques and translocation experiments [1]. PEs are deformable charged macromolecules and these two attributes are sufficient to make their mechanical behavior to be highly complex and investigating the aspects of their dynamics to be very challenging.…”

Section: Introductionmentioning

confidence: 99%

Polyelectrolytes polarization in nonuniform electric fields

Farahpour

Ejtehadi

Varnik

2014

Int. J. Mod. Phys. C

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Stretching dynamics of polymers in microfluidics is of particular interest for polymer scientists. As a charged polymer, a polyelectrolyte can be deformed from its coiled equilibrium configuration to an extended chain by applying uniform or non-uniform electric fields. By means of hybrid lattice Boltzmannmolecular dynamics simulations, we investigate how the condensed counterions around the polyelectrolyte contribute to the polymer stretching in inhomogeneous fields. As an application, we discuss the translocation phenomena and entropic traps, when the driving force is an applied external electric field.

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Beyond Gel Electrophoresis: Microfluidic Separations, Fluorescence Burst Analysis, and DNA Stretching

Cited by 176 publications

References 750 publications

Exploiting biased reptation for continuous flow preparative DNA fractionation in a versatile microfluidic platform

Exploiting biased reptation for continuous flow preparative DNA fractionation in a versatile microfluidic platform

Fast High-Throughput Screening of H1N1 Virus by Parallel Detection with Multichannel Microchip Electrophoresis

Polyelectrolytes polarization in nonuniform electric fields

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