Influence of Nucleotide-biased Fluorescence Emissions of SYBR Green II on the Result Consistence of Rolling Circle Amplification

Zhang, Bin; Jiang, Jiquan; Yuan, Ying; Guan, Yifu

doi:10.1007/s40242-019-9223-1

Cited by 3 publications

(3 citation statements)

References 23 publications

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“…Note that not only the DNA concentration is different but also the 180 nt DNA induces a higher signal intensity due to staining. However, the observed signal was <9% most likely because SYBR Green II tends to bind with a different affinity to the individual nucleotides as more recent research has shown . When 500 nM PNA was added to these samples, and the measurement was performed without a dye, the signals remained unchanged (Figure B).…”

Section: Resultsmentioning

confidence: 83%

“…However, the observed signal was <9% most likely because SYBR Green II tends to bind with a different affinity to the individual nucleotides as more recent research has shown. 35 When 500 nM PNA was added to these samples, and the measurement was performed without a dye, the signals remained unchanged ( Figure 4 B). This showed that PNAs are very sequence specific, even when a large amount of unspecific DNA is present in the sample.…”

Section: Resultsmentioning

confidence: 97%

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Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis

et al. 2023

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The space of advanced therapeutic modalities is currently evolving in rapid pace necessitating continuous improvement of analytical quality control methods. In order to evaluate the identity of nucleic acid species in gene therapy products, we propose a capillary electrophoresis-based gel free hybridization assay in which fluorescently labeled peptide nucleic acids (PNAs) are applied as affinity probes. PNAs are engineered organic polymers that share the base pairing properties with DNA and RNA but have an uncharged peptide backbone. In the present study, we conduct various proof-of-concept studies to identify the potential of PNA probes for advanced analytical characterization of novel therapeutic modalities like oligonucleotides, plasmids, mRNA, and DNA released by recombinant adeno-associated virus. For single-stranded nucleic acids up to 1000 nucleotides, the method is an excellent choice that proved to be highly specific by detecting DNA traces in complex samples, while having a limit of quantification in the picomolar range when multiple probes are used. For double-stranded samples, only fragments that are similar in size to the probe could be quantified. This limitation can be circumvented when target DNA is digested and multiple probes are used opening an alternative to quantitative PCR.

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 97%

Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis

et al. 2023

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“…Accurately quantifying the variants can be challenging, particularly in case of complex profiles or significant degradation (see Figure 4A, insert). The underlying cause for this is that the signal intensity is proportional to the mRNA size and intercalating dyes exhibit a specific affinity toward different nucleobases [33]. Furthermore, the laser-induced fluorescence (LIF) response can vary considerably within an analytical CE sequence.…”

Section: Characterization and Quantification Of Lnp Mrnamentioning

confidence: 99%

Electrophoretic characterization of LNP/AAV‐encapsulated nucleic acids: Strengths and weaknesses

Hutanu,

Ferreiro,

van Haasteren

et al. 2023

Electrophoresis

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The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra‐ and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non‐encapsulated or encapsulated large NAs (>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high‐performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.

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Signal-on electrochemical DNA (E-DNA) sensor for accurate quantification of nicking-assisted rolling circle amplification (N-RCA) products with attomolar sensitivity

Huang

et al. 2021

Anal. Methods

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Influence of Nucleotide-biased Fluorescence Emissions of SYBR Green II on the Result Consistence of Rolling Circle Amplification

Cited by 3 publications

References 23 publications

Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis

Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis

Electrophoretic characterization of LNP/AAV‐encapsulated nucleic acids: Strengths and weaknesses

Signal-on electrochemical DNA (E-DNA) sensor for accurate quantification of nicking-assisted rolling circle amplification (N-RCA) products with attomolar sensitivity

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