Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification

Chiminqgi, Mihelaiti; Moutereau, Stéphane; Pernet, Pascal; Conti, Marc; Barbu, Véronique; Lemant, Jerôme; Sacko, Mory; Vaubourdolle, Michel; Loric, Sylvain

doi:10.1515/cclm.2007.191

Cited by 21 publications

(23 citation statements)

References 8 publications

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“…Numerous investigators over the past two decades have turned to the use of OliGreen ® (OG) for single-stranded (ss) DNA and PicoGreen ® (PG) for double-stranded (ds) DNA detection and quantitation in buffer for quantitative polymerase chain reaction (QPCR) [3–6] and other buffer-based biosensor [7–11] and other assay applications [12–22]. OG and PG have been used to track and quantify ss and ds DNA in serum or plasma [15, 17–19] despite the high autofluorescence background of serum in the blue-green region of the spectrum [23] where these dyes emit, because these dyes can exhibit huge increases in fluorescence (e.g., a greater than 1,000-fold increase for PG [24]) when combined with DNA in solution.…”

Section: Introductionmentioning

confidence: 99%

Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum

Bruno

Sivils

2016

J Fluoresc

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Spectrofluorometric and emission peak titration and timed studies of OliGreen (OG) and PicoGreen (PG) were conducted in Tris EDTA (TE) buffer, pooled rat and fetal bovine serum with two different aptamers of 72 and 192 bases in length to determine if OG or PG were suitable for aptamer pharmacokinetic (PK) studies in sera. Results indicated that OG and PG detected the single-stranded (ss) and double- stranded (ds) stem-loop structures of the two aptamers quite well in TE with reliable standard curves having exponential character (or several linear detection regions) up to 1 μg/ml of aptamer DNA with detection limits of ~ 1 ng/ml. The intensity of OG and PG staining appeared to correlate with the number and percentage of ss and ds bases in each aptamer. OG and PG fluorescence in pooled rat serum or fetal bovine serum (FBS) did not titer as a function of DNA aptamer concentration from 1 μg/ml to 1 ng/ml. This lack of OG or PG aptamer assays in serum is contrary to most published reports of OG or PG assays for ss antisense oligonucleotides, ds PCR amplicons or other types of DNA in serum or plasma. Further studies suggested that the lack of OG and PG assay titration in serum might not be entirely due to aptamer degradation from nucleases in serum since the fluorescence signals in serum appeared relatively stable over time from 30 minutes to four hours. A hypothesis is presented which attributes the inability of OG or PG to assay aptamers in serum to a combination of high blue-green autofluorescence in serum with possible serum nuclease degradation of aptamers over time and the changing aptamer to serum protein ratio coupled to nonspecific binding of serum proteins to aptamers thereby possibly changing aptamer conformations as a function of aptamer concentration during titration experiments.

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Section: Introductionmentioning

confidence: 99%

Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum

Bruno

Sivils

2016

J Fluoresc

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“…Until recently the PCR (polymerase chain reaction) technique was exclusively employed for the purpose of detection of small quantities of specific DNA sequences in solution [1][2][3][4]. The power of PCR is in the ability to amplify DNA fragments to the level where simple, usually a fluorescent dye (e.g.…”

Section: Introductionmentioning

confidence: 99%

“…The power of PCR is in the ability to amplify DNA fragments to the level where simple, usually a fluorescent dye (e.g. PicoGreen, SYBR Green) [2] binding approaches, could easily register the presence of large amounts of amplified DNA.…”

Section: Introductionmentioning

confidence: 99%

5-Color Multiplexed Microwave-Accelerated Metal-Enhanced Fluorescence: Detection and Analysis of Multiple DNA Sequences from within one Sample Well within a Few Seconds

Dragan

Geddes

2014

J Fluoresc

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We present a potentially highly sensitive and selective bio-assay for the potential detection of any five different DNA sequences from one sample in one well. The assay is based on a DNA "rapid catch and signal" (DNA-RCS) technology developed for the detection of different DNA sequences from a sample well area. Our signal amplification utilizes the metal-enhanced fluorescence (MEF) of dyes attached to the probe-DNAs, which hybridizes with the pre-formed mixture of anchor-DNA scaffolds on silver island films (SiFs). Low-power microwave irradiation accelerates both the formation of the anchor-DNA scaffold on the SiF-surface and anchor/probe DNA hybridization, i.e. "rapid catch" of target DNAs from a bulk solution, decreasing the assay run time from hours to only a few seconds. Localization of signaling dye-labels close to the SiFs make them extremely photostable, which allows for collecting/integrating the signal over a long time period. To demonstrate a 5 color DNA assay (5-plex) we have used a range of readily available Alexa™ dyes. Advantages and perspectives of the RCS-technologies ability to detect 5 different DNA sequences from within one plate-well are discussed.

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“…The identification of living organisms and the detection of different microorganism mutations and strains of pathogenic bacteria, which cause severe diseases in humans is a challenging goal, and is the focus of much research today [1][2][3][4][5].…”

Section: Introductionmentioning

confidence: 99%

“…After amplification, DNA material can be easily detected by common analytical methods. Despite the obvious advantage of PCR in DNA detection, this approach has some disadvantages [2,8,9], e.g., sensitivity to DNA material contaminants; misreading; quite high cost of analysis, reagents, and time to fulfill experiments; and, most importantly, limited utility as a general fast and easy point-ofcare method of specific DNA sequence quantification [2,3].…”

Section: Introductionmentioning

confidence: 99%

Rapid Catch and Signal (RCS) Technology Platform: Multiplexed Three-Color, 30 s Microwave-Accelerated Metal-Enhanced Fluorescence DNA Assays

2014

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We present an innovative "Rapid Catch and Signal" (DNA-RCS) technology for the simultaneous highly selective detection of multiple specific DNA sequences in solution. The DNA-RCS technology combines advantages of microwave-accelerated DNA hybridization (Rapid Catch) with the metal-enhanced fluorescence (MEF) technology (Signal), to enable specific DNA's to be detected at high sensitivity within seconds. Fluorescent DNA labels, which play the role of molecular sensor probes, show a strong response upon DNA hybridization, due to fluorophore coupling with nanoparticle plasmons at a short (10-30 nm) distance from the surface. We have also shown that the fluorophore sensor probes demonstrate high photostability due to close proximity to a SiF surface, which significantly increases the total stability and reliability of the assay. Applications of the DNA-RCS technology in the life sciences, its advantages and benefits as compared to other DNA detection schemes, such as PCR, are subsequently discussed.

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Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification

Cited by 21 publications

References 8 publications

Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum

Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum

5-Color Multiplexed Microwave-Accelerated Metal-Enhanced Fluorescence: Detection and Analysis of Multiple DNA Sequences from within one Sample Well within a Few Seconds

Rapid Catch and Signal (RCS) Technology Platform: Multiplexed Three-Color, 30 s Microwave-Accelerated Metal-Enhanced Fluorescence DNA Assays

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