Fluorometric Assay Using Dimeric Dyes for Double- and Single-Stranded DNA and RNA with Picogram Sensitivity

Rye, Hays S.; Dabora, Jonathan M.; Quesada, Mark A.; Mathies, Richard A.; Glazer, Alexander N.

doi:10.1006/abio.1993.1020

Cited by 243 publications

(112 citation statements)

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“…Numerous materials, such as organic fluorescent dyes (4)(5)(6)(7)(8), semiconductor quantum dots (9,10) and luminescent metal-ligand complexes (11)(12)(13)(14), have been widely used as fluorescent probes for DNA detection. However, there are still some intrinsic limitations in these materials.…”

Section: Introductionmentioning

confidence: 99%

Luminescent and hydrophilic LaF₃–polymer nanocomposite for DNA detection

Wang

2008

Luminescence

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Luminescent LaF 3 -Ce 3+ /Tb 3+ nanocrystals have been successfully prepared via a simple wet chemical technique. For the next bioapplication, these nanoparticles dispersed in cyclohexane have also been functionalized with poly(Stco-MAA), based on a designed oil-in-water microemulsion system. These polymer-coated nanospheres are water-soluble and bioconjugable. Unlike semiconductor quantum dots, the as-prepared lanthanum fluoride nanocrystals possess nonsize-dependent emissions and completely stable photocycles. With functionalized LaF 3 nanospheres as fluorescence probes, a fluorescence method was developed for the rapid quantitative analysis of DNA, due to the quenching effect of fluorescence by the DNA. Under optimum conditions, the fluorescence intensity was proportional to the concentration of the introduced DNA over the range 2.5-35 mg/mL for calf thymus DNA (ctDNA) and 2.5-30 mg/mL for fish sperm DNA (fsDNA), respectively.

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Section: Introductionmentioning

confidence: 99%

Luminescent and hydrophilic LaF₃–polymer nanocomposite for DNA detection

Wang

2008

Luminescence

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“…The performance of these techniques is dependent on fluorochrome labels with a high sensitivity and high resistance to photobleaching. Numerous fluorescent probes, including magdala red (98,106), hypocrellin A (107), thiazole orange homodimer, oxazole yellow homodimer (77), and ethidium bromide (97), have been used for quantification of RNA. RiboGreen (Molecular Probes), another fluorescent probe for RNA quantification in solution, and arguably the most sensitive RNA probe available, allows concentrations as low as 1 ng ml Ϫ1 to be quantified with a standard spectrofluorimeter.…”

Section: Quantification Of Total Rna Concentrationsmentioning

confidence: 99%

Detection and Quantification of Gene Expression in Environmental Bacteriology

Sharkey¹,

Banat²,

Marchant³

2004

Appl Environ Microbiol

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“…These mixtures were loaded into a microtiter tray and scanned at 532 nm with the confocal scanning system at the same time as the gel. The scanner settings and the sampled volume were identical for the gel and the microtiter plate (12). Appropriate background corrections were appled to both sets of data.…”

Section: Quantitation Of Hsf Dtmentioning

confidence: 99%

Environment-sensitive Labels in Multiplex Fluorescence Analyses of Protein-DNA Complexes

Drees¹,

Rye²,

Glazer

et al. 1996

Journal of Biological Chemistry

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Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in gel-shifted complexes with specific DNA. A construct ( The "gel mobility shift" or "band-shift" assay is widely used to detect and analyze protein-DNA interactions. The most direct way to determine the stoichiometry of protein-DNA complexes resolved in such experiments is to measure independently the amounts of both DNA and protein in a band on a gel. Quantitation of DNA in the shifted complex is relatively straightforward with 32 P end-labeling, but accurate measurement of protein can be quite challenging. Protein quantities in shifted complexes have been measured in double-labeling experiments using Coomassie staining, Western blot, and radioisotopic labeling of the protein with 3 H, 125 I, and 35 S (1-7). Drawbacks of these methods include limitations on sensitivity, difficulty in ensuring accurately the specific activity of radiolabeled proteins, and the need to handle the gel after electrophoresis for staining or blotting, or to cut out gel bands for scintillation counting. Consequently, few accurate determinations of the molar ratios of protein-to-DNA have been reported.We have shown that multiplex fluorescence analysis is a superior alternative method of characterizing protein-DNA complexes (8). In our earlier work, we described the first detailed analysis of the application of fluorescent polycationic intercalating dyes with high affinity for double-stranded DNA to the study of protein-DNA interactions. Such intercalating dyes, in conjunction with high sensitivity laser-excited, confocal, fluorescence scanning systems, allow detection of doublestranded DNA in agarose or acrylamide gels with a sensitivity comparable to that of autoradiography (9 -12). We used such dyes in gel mobility shift assays to detect the multiple protein-DNA complexes formed by the heat shock transcription factor (HSF) 1 (8,13,14). Using a truncation of Kluyveromyces lactis HSF that contains the DNA-binding and trimerization domains (HSF DT ), we were able to detect fluorescent dye-labeled HSF DT -DNA complexes with a spatial resolution superior to that of conventional autoradiography, and therefore we were able to analyze multimer protein-DNA complexes that are not resolved by traditional methods. An analysis of the mobilities of the multiple HSF DT -DNA complexes, indicated that HSF forms multimeric complexes on DNA by the addition of trimeric units (8).To measure the absolute quantities of protein and DNA in each complex, we developed a two-color mobility shift fluorescence assay with a mutant HSF DT engineered for site-specific labeling with fluorescein and target DNA labeled with thiazole orange-thiazole blue heterodimer (TOTAB), an "energy transfer" dye (15, 16). A technical difficulty was enco...

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Fluorometric Assay Using Dimeric Dyes for Double- and Single-Stranded DNA and RNA with Picogram Sensitivity

Cited by 243 publications

References 0 publications

Luminescent and hydrophilic LaF₃–polymer nanocomposite for DNA detection

Luminescent and hydrophilic LaF₃–polymer nanocomposite for DNA detection

Detection and Quantification of Gene Expression in Environmental Bacteriology

Environment-sensitive Labels in Multiplex Fluorescence Analyses of Protein-DNA Complexes

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Fluorometric Assay Using Dimeric Dyes for Double- and Single-Stranded DNA and RNA with Picogram Sensitivity

Cited by 243 publications

References 0 publications

Luminescent and hydrophilic LaF3–polymer nanocomposite for DNA detection

Luminescent and hydrophilic LaF3–polymer nanocomposite for DNA detection

Detection and Quantification of Gene Expression in Environmental Bacteriology

Environment-sensitive Labels in Multiplex Fluorescence Analyses of Protein-DNA Complexes

Contact Info

Product

Resources

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Luminescent and hydrophilic LaF₃–polymer nanocomposite for DNA detection

Luminescent and hydrophilic LaF₃–polymer nanocomposite for DNA detection