High-Throughput Analysis With 96-Capillary Array Electrophoresis and Integrated Sample Preparation for DNA Sequencing Based on Laser Induced Fluorescence Detection

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The purpose of this research was to improve the fluorescence detection for the multiplexed capillary array electrophoresis, extend its use beyond the genomic analysis, and to develop an integrated micro-sample preparation system for high-throughput DNA sequencing. The authors first demonstrated multiplexed capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations in a 96-capillary array system with laser-induced fluorescence detection. Migration times of four kinds of fluoresceins and six polyaromatic hydrocarbons (PAHs) are normalized to one of the capillaries using two internal standards. The relative standard deviations (RSD) after normalization are 0.6-1.4% for the fluoresceins and 0.1-1.5% for the PAHs. ... continued below

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3477 Kilobytes pages

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Xue, Gang December 31, 2001.

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  • Ames Laboratory
    Publisher Info: Ames Lab., IA (United States)
    Place of Publication: Iowa

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The purpose of this research was to improve the fluorescence detection for the multiplexed capillary array electrophoresis, extend its use beyond the genomic analysis, and to develop an integrated micro-sample preparation system for high-throughput DNA sequencing. The authors first demonstrated multiplexed capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations in a 96-capillary array system with laser-induced fluorescence detection. Migration times of four kinds of fluoresceins and six polyaromatic hydrocarbons (PAHs) are normalized to one of the capillaries using two internal standards. The relative standard deviations (RSD) after normalization are 0.6-1.4% for the fluoresceins and 0.1-1.5% for the PAHs. Quantitative calibration of the separations based on peak areas is also performed, again with substantial improvement over the raw data. This opens up the possibility of performing massively parallel separations for high-throughput chemical analysis for process monitoring, combinatorial synthesis, and clinical diagnosis. The authors further improved the fluorescence detection by step laser scanning. A computer-controlled galvanometer scanner is adapted for scanning a focused laser beam across a 96-capillary array for laser-induced fluorescence detection. The signal at a single photomultiplier tube is temporally sorted to distinguish among the capillaries. The limit of detection for fluorescein is 3 x 10{sup -11} M (S/N = 3) for 5-mW of total laser power scanned at 4 Hz. The observed cross-talk among capillaries is 0.2%. Advantages include the efficient utilization of light due to the high duty-cycle of step scan, good detection performance due to the reduction of stray light, ruggedness due to the small mass of the galvanometer mirror, low cost due to the simplicity of components, and flexibility due to the independent paths for excitation and emission.

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3477 Kilobytes pages

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OSTI as DE00803101

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  • Other Information: TH: Thesis (Ph.D.); Submitted to Iowa State Univ., Ames, IA (US)

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  • Report No.: IS-T 1992
  • Grant Number: W-7405-Eng-82
  • Office of Scientific & Technical Information Report Number: 803101
  • Archival Resource Key: ark:/67531/metadc735382

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Office of Scientific & Technical Information Technical Reports

Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

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  • December 31, 2001

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  • Oct. 19, 2015, 7:39 p.m.

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  • Nov. 11, 2015, 6:41 p.m.

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Xue, Gang. High-Throughput Analysis With 96-Capillary Array Electrophoresis and Integrated Sample Preparation for DNA Sequencing Based on Laser Induced Fluorescence Detection, thesis or dissertation, December 31, 2001; Iowa. (digital.library.unt.edu/ark:/67531/metadc735382/: accessed July 16, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.