Radial Capillary Array Electrophoresis Microplate and Scanner for High-Performance Nucleic Acid Analysis

Shi, Yining; Simpson, Peter C.; Scherer, James R.; Wexler, David; Skibola, Christine F.; Smith, Martyn T.; Mathies, Richard A.

doi:10.1021/ac990518p

Cited by 284 publications

(285 citation statements)

References 53 publications

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“…The Berkeley 4-color rotary scanner has been described in detail (18). A conceptual schematic of the system is presented in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Our group has pioneered the design and construction of microfabricated capillary array electrophoresis devices demonstrating rectilinear networks of 12 and 48 channels for parallel DNA fragment analysis (16,17). The field was further advanced with the presentation of a 96-lane radial microchannel plate (MCP) layout together with a rotary confocal scanning detector and its use for high throughput genotyping (18).…”

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confidence: 99%

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High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor

Paegel

Emrich

Wedemayer

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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249

179

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High throughput DNA sequencing has been performed by using a microfabricated 96-channel radial capillary array electrophoresis ( CAE) microchannel plate detected by a 4-color rotary confocal fluorescence scanner. The microchannel plate features a novel injector for uniform sieving matrix loading as well as high resolution, tapered turns that provide an effective separation length of 15.9 cm on a compact 150-mm diameter wafer. Expanded common buffer chambers for the cathode, anode, and waste reservoirs are used to simplify electrode addressing and to counteract buffering capacity depletion arising from the high electrophoretic current. DNA sequencing data from 95 successful lanes out of 96 lanes run in parallel were batch-processed with BASEFINDER, producing an average read length of 430 bp (phred q > 20). Phred quality values were found to exceed 40 (0.01% probability of incorrectly calling a base) for over 80% of the read length. The CAE system demonstrated here produces sequencing data at a rate of 1.7 kbp͞min, a 5-fold increase over current commercial capillary array electrophoresis technology. Additionally, this system permits lower reagent volumes and lower sample concentrations, and it presents numerous possibilities for integrated sample preparation and handling. The unique capabilities of CAE technology should make it the next generation, high performance DNA sequencing platform.A dvances in DNA sequencing technologies over the past decade have been driven by the Human Genome Project (1). To achieve the ambitious goals of the genome project, sequencing centers replaced slab gel-based systems with capillary array electrophoresis (CAE) instruments that are based either on the scanning confocal detector developed in our laboratory (2, 3) or on sheath-flow detection (4, 5). Although the draft sequence of the human genome is now ''complete'' (6), expanded sequencing capacity is still required for the characterization of human sequence variation and for sequencing other genomes. In pursuing these goals, many technology development efforts are focused on the use of microfabricated electrophoretic analysis systems.Microfabricated capillary electrophoresis devices were introduced by Manz and coworkers (7,8) in the early 1990s. These microchips efficiently and very rapidly separated mixtures of fluorescent dyes and fluorescently labeled amino acids by using a fraction of the conventionally required capillary length. Initial experiments were rapidly followed by the demonstration of protocols for rapid DNA sizing and sequencing (9, 10), integrated thermal cycling fluidic circuitry (11), and clinically relevant genotyping (12). In subsequent work, the classical variables of DNA sequencing separations such as column length, sieving matrix composition, injection parameters, etc. have been explored and optimized (13,14), producing read lengths as long as 800 bp for a 40-cm-long microfabricated capillary in 80 min (15).Microfabricated capillary array electrophoresis ( CAE) devices are advantageous compared with drawn cap...

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“…The Berkeley 4-color rotary scanner has been described in detail (18). A conceptual schematic of the system is presented in Fig.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor

Paegel

Emrich

Wedemayer

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

249

179

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“…Multichannel structures for parallel processing were introduced with straight [31,40] as well as with radial channels [78]. For postcolumn derivatization chip configuration, an extra side channel was connected to the end of the separation column [79].…”

Section: Microchip Layout Considerationsmentioning

confidence: 99%

New advances in microchip fabrication for electrochromatography

Szekeres

Guttman

2005

Electrophoresis

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There is a great demand in separation technologies for faster and more effective analysis processes. Miniaturization is a suitable technique for satisfying this demand as reduction in size gives increased separation speed with higher efficiency. CEC is an electric-field-mediated separation technique where the liquid flow is generated by the electric field itself. The main advantage of using electric field over pressure for flow generation is the flat flow profile of the EOF; thus, CEC is one of the best candidates to construct a novel and high-efficiency microanalytical device. The aim of the present paper is to review the basic fabrication and bonding principles, as well as connection and system integration options for microfluidics-based electrochromatography. The physical structure and fluidic channel formation are critically evaluated, including glass microstructuring and fusion bonding. Recent developments in nanoflow measurements and the application of various flow control units are also extensively discussed.

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“…Many techniques developed in traditional CE have been transferred onto microchip CE with appropriate modifications [3][4][5][6][7]. Multilane microchips have been developed to obtain high throughput analysis and were applied in DNA sequencing, genotyping, pharmaceuticals and other areas [8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Frontal analysis in microchip CE: A simple and accurate method for determination of protein–DNA dissociation constant

et al. 2007

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Equilibrium constants, such as the dissociation constant (K d ), are a key measurement of noncovalent interactions that are of importance to the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate capillary electrophoresis (CE) method for the determination of K d . Microchip CE coupled with laser-induced fluorescence (LIF) detection was used to determine K d of protein-DNA interactions using the FA method. A model system of immunoglobulin E (IgE) and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The K d of the IgE-aptamer pair was determined as 6 ± 2 nM which is consistent with the reported results (8 nM).

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Radial Capillary Array Electrophoresis Microplate and Scanner for High-Performance Nucleic Acid Analysis

Cited by 284 publications

References 53 publications

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor

New advances in microchip fabrication for electrochromatography

Frontal analysis in microchip CE: A simple and accurate method for determination of protein–DNA dissociation constant

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