A Quantitative Fluorescence-Based Microplate Assay for the Determination of Double-Stranded DNA Using SYBR Green I and a Standard Ultraviolet Transilluminator Gel Imaging System

Vitzthum, Frank; Geiger, G.; Bisswanger, Hans; Brunner, Herwig; Bernhagen, Jürgen

doi:10.1006/abio.1999.4298

Cited by 125 publications

(86 citation statements)

References 14 publications

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“…of DNA (33,37). These effects were not apparent, however, in the present study and seemed not to impair the applicability of SYBR green I for sensitive biomass determinations.…”

contrasting

confidence: 56%

“…However, the different dyes were found to vary remarkably in terms of sensitivity and the range of cell numbers they can detect. Cyanine dyes (PicoGreen and SYBR green) that are commonly employed for the quantification of dissolved DNA (20,33,37) were the most sensitive and allowed reliable quantification of microbial cells. Consistent with this, PicoGreen has previously been used for the determination of microbial biomass in environmental samples (14,31).…”

Section: Discussionmentioning

confidence: 99%

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Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

Martens‐Habbena

Sass

2006

Appl Environ Microbiol

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The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, flow cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial cells in cultures and most-probable-number series. The assay combines fluorescent nucleic acid staining and subsequent fluorescence measurement in suspension. Six different fluorescent dyes (acridine orange, DAPI [4,6-diamidino-2-phenylindole], ethidium bromide, PicoGreen, and SYBR green I and II) were evaluated. SYBR green I was found to be the most sensitive dye and allowed the quantification of 50,000 to up to 1.5 ؋ 10 8 Escherichia coli cells per ml sample. The rapid staining procedure was robust against interference from rRNA, sample fixation by the addition of glutaric dialdehyde, and reducing agents such as sodium dithionite, sodium sulfide, and ferrous sulfide. It worked well with phylogenetically distant bacterial and archaeal strains. Excellent agreement with optical density measurements of cell increases was achieved during growth experiments performed with aerobic and sulfate-reducing bacteria. The assay offers a time-saving, more sensitive alternative to epifluorescence microscopy analysis of most-probable-number dilution series. This method simplifies the quantification of microbial cells in pure cultures as well as enrichments and is particularly suited for low cell densities.

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“…of DNA (33,37). These effects were not apparent, however, in the present study and seemed not to impair the applicability of SYBR green I for sensitive biomass determinations.…”

contrasting

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

Martens‐Habbena

Sass

2006

Appl Environ Microbiol

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“…Single-stranded DNA and RNA are also detected, although at approximately 10-fold lower sensitivities (14). It is typically used in gel electrophoresis methods and is 50 to 100 times more sensitive than ethidium bromide as a nucleic acid-specific fluorochrome.…”

Section: Resultsmentioning

confidence: 99%

Simple and Inexpensive Fluorescence-Based Technique for High-Throughput Antimalarial Drug Screening

Smilkstein

Sriwilaijaroen

Kelly³

et al. 2004

Antimicrob Agents Chemother

1,033

945

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Radioisotopic assays involve expense, multistep protocols, equipment, and radioactivity safety requirements which are problematic in high-throughput drug testing. This study reports an alternative, simple, robust, inexpensive, one-step fluorescence assay for use in antimalarial drug screening. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. A side-by-side comparison of this fluorescence assay and a standard radioisotopic method was performed by testing known antimalarial agents against Plasmodium falciparum strain D6. Both assay methods were used to determine the effective concentration of drug that resulted in a 50% reduction in the observed counts (EC 50 ) after 48 h of parasite growth in the presence of each drug. The EC 50 s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis--(N,N-diethylamino)-amyloxyxanthone were similar or identical by both techniques. The results obtained with this new fluorescence assay suggest that it may be an ideal method for highthroughput antimalarial drug screening.The global scope of malaria and the spread of drug-resistant Plasmodium falciparum make the need for improved therapy undeniable (4). Assessment of both existing drugs and new antimalarials, alone or in combination, requires reliable methods for high-throughput testing. For decades, antimalarial drug effects have been measured in vitro by quantifying parasite uptake of radioactive substrates as a measure of growth and viability in the presence of the test drug (2, 3). [3 H]hypoxanthine is the most widely used radiolabel, but because it requires purine starvation prior to the assay, the [ 3 H]ethanolamine assay is favored by our laboratory. While these methods are accurate and reliable, they rely on relatively expensive radioisotopes and multistep procedures that become increasingly problematic and impractical as the volume of testing increases. We report on the development of an alternative, fluorescencebased in vitro assay for use for the high-throughput screening of the activities of drugs against P. falciparum. The goals for the assay included simplicity, cost savings, robust performance, applicability to automated analysis, and speed.The principle behind the assay is the contrast between host erythrocytes, which lack DNA and RNA, and the malaria parasites, which do not and which are thus readily stained with dyes that show enhanced fluorescence in the presence of nucleic acids (8). Fluorescence-based in vitro antimalarial assays have been developed previously (1, 12) but have required complex, multistep protocols or additional equipment not amenable to rapid, high-throughput use. This report establishes and validates a facile, one-step method that uses the same principle. MATERIALS AND METHODSChloroquine diphosphate, quinine sulfate, artemisinin, and saponin were purchased from Sigma Aldrich Chemical Company (St. Louis, Mo.); mefloquine was obtained from Walter Reed Army Institute for Research (Silver Spring, Md.); and 3,6-b...

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“…These results revealed that potassium phosphate solution had stronger elution ability at higher pH value. However, the elution peak height at pH 11.0 decreased sharply, which may be ascribed to the decrease in the fluorescence intensity of Sybr green I at this pH [30]. DNA was also reported unstable at extremely high pH value [31].…”

Section: Optimization Of the Extraction Conditionsmentioning

confidence: 95%

Extraction of genomic DNA using a new amino silica monolithic column

Liu

Yang

et al. 2009

J of Separation Science

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Extraction of genomic DNA using a new amino silica monolithic column A new amino silica monolithic column was developed for DNA extraction in a miniaturized format. The monolithic column was prepared in situ by polymerization of tetraethoxysilane (TEOS) and N-(b-aminoethyl)-c-aminopropylmethyldimethoxysilane (AEAPMDMS). DNA was loaded in 50 mM tris(hydroxylmethyl)aminomethane -EDTA buffer at pH 7.0 and eluted with 300 mM potassium phosphate solution at pH 10.0. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 56 ng DNA with an extraction efficiency of 71 l 5.2% (X l RSD). When the amino silica monolithic column was applied to extract genomic DNA from the whole blood of crucian carp, an extraction efficiency of 52 l 5.6% (X l RSD) was obtained by three extractions. Since the chaotropic-based sample loading and organic solvent wash steps were avoided in this procedure, the purified DNA was suitable for downstream processes such as PCR. This amino silica monolithic column was demonstrated to allow rapid and efficient DNA purification in microscale.

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A Quantitative Fluorescence-Based Microplate Assay for the Determination of Double-Stranded DNA Using SYBR Green I and a Standard Ultraviolet Transilluminator Gel Imaging System

Cited by 125 publications

References 14 publications

Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

Simple and Inexpensive Fluorescence-Based Technique for High-Throughput Antimalarial Drug Screening

Extraction of genomic DNA using a new amino silica monolithic column

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