Mono‐ and bis‐intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis

McIntosh, Sara L.; Deligeorgiev, Todor; Gadjev, Nikolai; McGown, Linda B.

doi:10.1002/1522-2683(200205)23:10<1473::aid-elps1473>3.0.co;2-#

Cited by 8 publications

(2 citation statements)

References 18 publications

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“…The eTag reporter consists of the eTag molecule along with the 5 0 terminal nucleic acid residue cleaved from a signal probe by specific enzymatic reaction (vide infra). It is well documented that CE is a powerful way to separate fluorescent molecules or DNA fragments, such as DNA fragments from sequencing reactions (21)(22)(23), restriction digestions (24,25), multiplex PCR assay (26) or from ligation assays (27). Similarly, CE can also be applied in separating and quantifying eTag reporters, since each reporter possesses a unique electrophoretic mobility.…”

Section: Resultsmentioning

confidence: 99%

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

Tian¹

2004

Nucleic Acids Research

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We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1beta. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise, as evaluated by measuring qualified Z' factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection and the isothermal linear amplification characteristics of the Invader assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.

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Section: Resultsmentioning

confidence: 99%

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

Tian¹

2004

Nucleic Acids Research

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“…The present work is a departure from previous applications of uorescence lifetime detection in chemical separations to well-de ned analytical schemes, such as multiplex detection in DNA sequencing, [26][27][28] DNA ngerprinting, 29,30 and detection of simple mixtures of compo und s, 3 1 -3 4 in its fo cus on a h ig hly com p lex, polydisperse system of macromolecules. Since uorescence lifetimes of the intrinsic uorophores will be sensitive to the m icroenvironments of the uorophores, detection and resolution of the uorescence lifetimes associated with the various subfractions of humic molecules as they migrate in the ''humic hump'' in CZE should provide insight into the polydispersity, heterogeneity, aggregation, complexation, and binding interactions of humic substances; the effects of extrinsic factors, such as pH, ionic strength, metals, detergents, and organic compounds, on these properties could then be studied.…”

Section: Introductionmentioning

confidence: 98%

On-the-Fly Fluorescence Lifetime Detection of Humic Substances in Capillary Electrophoresis

Hewitt

McGown

2003

Appl Spectrosc

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On-the-fly fluorescence lifetime detection was investigated as a tool for studying humic substances in capillary zone electrophoresis (CZE). Humic substances are complex, heterogeneous mixtures of natural products that tend to migrate in a single, broad CZE peak. The intrinsic fluorescence lifetime of five humic substances from the International Humic Substances Society (IHSS) was monitored using excitation at 488 or 364 nm to produce intensity-lifetime electropherograms for each of the substances. Each frequency-domain lifetime measurement, collected at subsecond intervals during the CZE run, contains the equivalent of a complete decay profile. Lifetime analysis of each decay profile was used to construct a lifetime-resolved electropherogram for each lifetime component, from which the variation in relative intensity contributions of each lifetime across the broad CZE peak could be determined. Absorption spectra, fluorescence excitation-emission spectra, and lifetime profiles of batch solutions of the samples were determined as well. It was found that, whereas absorption and fluorescence spectral characteristics tended to discriminate between humic acids and fulvic acids, the batch solution lifetime profiles discriminated instead between samples from different sources, regardless of fraction. On-the-fly lifetime detection provided a more detailed view of the fluorescence decay of the samples, including greater resolution of lifetimes for two of the fulvic acids and greater discrimination among samples based on lifetime profiles across the CZE peaks.

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Separation of double‐stranded DNA fragments by capillary electrophoresis: Impacts of poly(ethylene oxide), gold nanoparticles, ethidium bromide, and pH

2004

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The separation of DNA by capillary electrophoresis using poly(ethylene oxide) (PEO) containing gold nanoparticles (GNPs) is presented. The impacts of PEO, GNPs, ethidium bromide (EtBr), and pH on the separation of double-stranded DNA have been carefully explored. Using a capillary dynamically coated with 5.0% poly(vinylpyrrolidone) and filled with 0.2% PEO containing 0.3 x GNPs (the viscosity less than 15 cP), we have demonstrated the separation of DNA markers V and VI within 5 min at pH 8.0 and 9.0. In terms of resolution and reproducibility, GNPs have a greater impact on the separation of DNA at pH 9.0. Resolution improvements for large DNA fragments (> 300 base pairs, bp) are greater than those for small ones in the presence of GNPs. It is important to point out that reproducibility is excellent (relative standard deviations for the migration times less than 0.5%) and thus no further dynamic coating is required in at least 20 consecutive runs in the presence of GNPs. Using 0.2% PEO (pH 9.0) containing 0.3 x GNPs, the separation of DNA fragments ranging in size from 21 to 23,130 bp was accomplished in 7 min. The results presented in this study show the advantage of PEO containing GNPs for DNA separation, including rapidity, high resolving power, excellent reproducibility, and ease of filling capillaries.

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Mono‐ and bis‐intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis

Cited by 8 publications

References 18 publications

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis

On-the-Fly Fluorescence Lifetime Detection of Humic Substances in Capillary Electrophoresis

Separation of double‐stranded DNA fragments by capillary electrophoresis: Impacts of poly(ethylene oxide), gold nanoparticles, ethidium bromide, and pH

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