High-speed separations of DNA sequencing reactions by capillary electrophoresis

Drossman, Howard; Luckey, John A.; Kostichka, Anthony J.; D’Cunha, Jonathan; Smith, Lloyd M.

doi:10.1021/ac00208a003

Cited by 260 publications

(107 citation statements)

References 12 publications

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“…For many years, slab gel systems have been used as the workhorse for gene mapping, DNA sequencing, and disease diagnosis. Recently, advances such as thin slab gels (1), capillary electrophoresis (CE) (2)(3)(4)(5), and capillary array electrophoresis (CAE) (6)(7)(8)(9)(10)(11)(12) have significantly increased the performance of gel electrophoresis. However, to meet the growing demand for increased speed and sample throughput, more revolutionary approaches are needed.…”

mentioning

confidence: 99%

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates

Simpson

Roach

Woolley

et al. 1998

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

266

206

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Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal f luorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and f luorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.

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mentioning

confidence: 99%

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates

Simpson

Roach

Woolley

et al. 1998

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

266

206

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“…The sensitivity in the detection of FITC-labeled DNA is somewhat worse than those given in a published paper. 24 The major reason is attributed to the fact that no investigation was performed in this study to improve the sensitivity, e.g. reduction of scattered light and unwanted fluorescence from a capillary and a buffer solution.…”

Section: Semiconductor Lifmentioning

confidence: 99%

Detection of a Specific DNA Fragment by Free-Solution Capillary Electrophoresis Using a Fluorescent DNA Probe Measured by Semiconductor Laser-Induced Fluorometry

1996

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Semiconductor laser-induced fluorescence detection of native (target) DNA has been accomplished by free-solution capillary electrophoresis (CE) using an oligonucleotide labeled with a cyanine dye as a DNA probe. A mixture of probe DNA and target DNA, which is complementary to probe DNA, is incubated to form hybrid DNA; the mixture is then injected into a capillary. Single-stranded probe DNA and double-stranded hybrid DNA were separated by free-solution CE. A 19mer oligonucleotide labeled with fluorescein isothiocyanate (FITC) was used as probe DNA in a preliminary study. The stability of double-stranded DNA during migration was evaluated by changing the temperature of the solution in the capillary, i.e. the applied voltage in CE. The dissociation of double-stranded DNA was appreciable at high applied voltages. Thus, suppressing the temperature in the capillary, i.e. optimization of the voltage, was required in order to prevent the dissociation of double-stranded DNA. Furthermore, a new labeling reagent, a cyanine derivative, was synthesized in order to be applied to semiconductor laser-induced fluorometry. The detection limit was 8X 10-9 M for probe DNA.

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“…Many authors have provided excellent reviews [4][5][6][7][8][9][10] concerning the practice of CE in such a field. In earlier days, a cross-linked polyacrylamide gel was employed as the sieving matrix to support the size-dependent separation mechanism, termed capillary gel electrophoresis (CGE), 11,12 which could offer a resolution of a single base pair (bp) ranged from 15 bp to 500 bp. However, difficulties in preparing a capillary gel column, such as the formation of a bubble and the low duration of high voltage, have bottlenecked its further development.…”

Section: Introductionmentioning

confidence: 99%

Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution

Liu

Xie

2001

ANAL. SCI.

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It was demonstrated that a capillary electrophoresis (CE) method with a non-gel sieving solution has been developed to identify the orientation of DNA fragments in recombinant plasmids in molecular biology. The influences of the concentration of sieving polymer HEC, the applied electric field strength and sampling on CE separation were analyzed concerning the optimization of separation. YO-PRO-1 was used as a DNA intercalating reagent to facilitate fluorescence detection. Under the chosen conditions (buffer, 1 × TBE containing 1 µM YO-PRO-1 and 1.2% HEC; applied electric field strength, 200 V/cm; electrokinetic sampling: time, 5 s; voltage, -6 kV), three DNA markers (φ174/HaeIII, pBR322/HaeIII and λDNA/HindIII) were tested for further evaluating the relationship between the DNA size and the mobility. The established CE method conjugated with the enzymatic approach was successfully applied to identifying the DNA orientation of recombinant plasmid in transgene operations of a newly cloned gene from Arabidopsis Thaliana.

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High-speed separations of DNA sequencing reactions by capillary electrophoresis

Cited by 260 publications

References 12 publications

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates

High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates

Detection of a Specific DNA Fragment by Free-Solution Capillary Electrophoresis Using a Fluorescent DNA Probe Measured by Semiconductor Laser-Induced Fluorometry

Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution

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