Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath-flow cuvette

Crabtree, H. John; Bay, Sue; Lewis, Darren; Zhang, Jianzhong; Coulson, Larry D.; Fitzpatrick, Glen; Delinger, Scott L.; Harrison, David; Dovic̀hi, Norman J.

doi:10.1002/(sici)1522-2683(20000401)21:7<1329::aid-elps1329>3.0.co;2-2

Cited by 45 publications

(14 citation statements)

References 30 publications

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“…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.…”

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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.

248

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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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.

248

179

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“…However, this separation is performed without operator intervention or supervision. We have demonstrated that a multiple capillary sheath-flow cuvette is an efficient detector in high throughput DNA sequencing (22)(23)(24). We plan to modify the two-dimensional electrophoresis instrument so that 96 samples can be analyzed in parallel.…”

Section: Discussionmentioning

confidence: 99%

Fully Automated Two-dimensional Capillary Electrophoresis for High Sensitivity Protein Analysis

Michels

Schoenherr

et al. 2002

Molecular & Cellular Proteomics

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118

114

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We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a twodimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity. The resolution of complex samples into components requires sophisticated technology. Most separation techniques are capable of resolving, at most, several dozen components. Cal Giddings (1) recognized that the combination of two separation techniques is important in the resolution of complex mixtures. If the two separation techniques are based on unrelated characteristics of the sample, then the number of resolution elements is given by the product of the resolution elements of both separation steps. For example, isoelectric focusing and SDS-polyacrylamide gel electrophoresis can, individually, resolve ϳ50 components in a protein sample. Their combination, in two-dimensional electrophoresis, can resolve several thousand components (2).Unfortunately, classic two-dimensional electrophoresis requires manual manipulation of the sample. These manipulations, although reasonable for occasional use, are very tedious when performed in large-scale protein analysis projects. Furthermore, detection sensitivity is limited, and relatively large amounts of sample are required to detect weakly expressed components.Column-switching technology is an alternative means of performing two-dimensional separations and is commonly used in chromatographic resolution of complex samples. Most simply, a fraction containing the compound of interest is captured as it elutes from the first column and is transferred to another chromatographic column for additional resolution; fractionation is repeated with different chromatographic methods to achieve the desired purity.In multidimensional chromatography, also known as comprehensive chromatography, a second column is used to sequentially separate all fractions from the first column. These two-dimensional techniques are particularly useful when characterizing extremely complex samples. An early report used two successive chromatographic steps to purify peptides generated from the proteolytic digest of a human immunoglobulin (3).Jorgenson and others (4 -12) have developed elegant...

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“…There are two instruments that can rely on a sheath-flow cuvette to simultaneously monitor fluorescence from a linear array of capillaries. These instruments were developed by Kambara and Takahashi 33 at Hitachi and by Dovichi et al 34,35 at the University of Alberta. The sheath-flow cuvette CAE instrument has been licensed to Applied Biosystems and commercialized as "Model 3700 DNA sequencer".…”

Section: ·4 Capillary Array Electrophoresismentioning

confidence: 99%

The Human Genome Project: the Role of Analytical Chemists

Thangadurai¹

2004

ANAL. SCI.

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Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath-flow cuvette

Cited by 45 publications

References 30 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

Fully Automated Two-dimensional Capillary Electrophoresis for High Sensitivity Protein Analysis

The Human Genome Project: the Role of Analytical Chemists

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