A platform method for plasmid isoforms analysis by capillary gel electrophoresis

Wang, Meng; Liu, Jun‐Kai; Gao, Tie; Xu, Ling‐Li; Zhang, Xiaoxia; Nie, Jianhui; Li, Yan; Chen, Hong‐Xu

doi:10.1002/elps.202100343

Cited by 10 publications

(12 citation statements)

References 17 publications

(23 reference statements)

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“…Importantly, the almost topology-independent binding of DNA intercalators above their K d is advantageous for assays that aim to quantitatively compare different DNA topologies, e.g. to monitor the products of integration or topoisomerization reactions ( 61 , 65 , 91 , 92 ). In particular, for SYBR Gold DNA staining is essentially unbiased by topology for dye concentrations in the range of 1–2 μM, which is the concentration range that we previously identified as optimal for achieving a linear relationship between the fluorescence signal and the amount of DNA present ( 19 ).…”

Section: Resultsmentioning

confidence: 99%

Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments

Kolbeck,

Tišma,

Analikwu

et al. 2023

Nucleic Acids Research

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DNA stores our genetic information and is ubiquitous in applications, where it interacts with binding partners ranging from small molecules to large macromolecular complexes. Binding is modulated by mechanical strains in the molecule and can change local DNA structure. Frequently, DNA occurs in closed topological forms where topology and supercoiling add a global constraint to the interplay of binding-induced deformations and strain-modulated binding. Here, we present a quantitative model with a straight-forward numerical implementation of how the global constraints introduced by DNA topology modulate binding. We focus on fluorescent intercalators, which unwind DNA and enable direct quantification via fluorescence detection. Our model correctly describes bulk experiments using plasmids with different starting topologies, different intercalators, and over a broad range of intercalator and DNA concentrations. We demonstrate and quantitatively model supercoiling-dependent binding in a single-molecule assay, where we directly observe the different intercalator densities going from supercoiled to nicked DNA. The single-molecule assay provides direct access to binding kinetics and DNA supercoil dynamics. Our model has broad implications for the detection and quantification of DNA, including the use of psoralen for UV-induced DNA crosslinking to quantify torsional tension in vivo, and for the modulation of DNA binding in cellular contexts.

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Section: Resultsmentioning

confidence: 99%

Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments

Kolbeck,

Tišma,

Analikwu

et al. 2023

Nucleic Acids Research

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“…In one case, DNA as long as four megabase pairs could be observed (37). Capillary gel sieving resolves DNA structures by topology (39,40). This is important for plasmid-based biopharmaceuticals because supercoiled DNA plasmids are more effective for transfection.…”

Section: Nucleic Acidsmentioning

confidence: 99%

Gels in Microscale Electrophoresis

Holland

Casto-Boggess

2023

Annual Rev. Anal. Chem.

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Gel matrices are fundamental to electrophoresis analyses of biopolymers in microscale channels. Both capillary gel and microchannel gel electrophoresis systems have produced fundamental advances in the scientific community. These analytical techniques remain as foundational tools in bioanalytical chemistry and are indispensable in the field of biotherapeutics. This review summarizes the current state of gels in microscale channels and provides a brief description of electrophoretic transport in gels. In addition to the discussion of traditional polymers, several nontraditional gels are introduced. Advances in gel matrices highlighted include selective polymers modified to contain added functionality as well as thermally responsive gels formed through self-assembly. This review discusses cutting-edge applications to challenging areas of discovery in DNA, RNA, protein, and glycan analyses. Finally, emerging techniques that result in multifunctional assays for real-time biochemical processing in capillary and three-dimensional channels are identified.

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“…Capillary electrophoresis is widely used in the analysis of biological products due to its high resolution, multiple separation modes, less sample and reagent consumption, and high degree of automation [23]. Capillary gel electrophoresis (CGE) is a powerful tool used in the analysis of various nucleic acids, including plasmid [24], antisense oligonucleotide, small interfering ribonucleic acid, and messenger RNA (mRNA) [25]. CGE with laser‐induced fluorescence detector (LIF) can provide even better sensitivity and resolution via nucleic acids labeling [26].…”

Section: Introductionmentioning

confidence: 99%

“…CGE with laser‐induced fluorescence detector (LIF) can provide even better sensitivity and resolution via nucleic acids labeling [26]. DNA dyes frequently used in CGE‐LIF analysis were SYBR Gold [24], GelGreen [26], and SYBR Green I [27]. These dyes have different staining efficiencies, of which SYBR Gold exhibited prominent performance in terms of sensitivity and cost‐effectiveness.…”

Section: Introductionmentioning

confidence: 99%

Size distribution analysis of residual host cell DNA fragments in lentivirus by CGE‐LIF

et al. 2022

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During the production of cell and gene therapy products, residual host cell DNA (HCD) could cause safety risks of the biological products, and the longer the residual HCD fragment, the greater the risk to the human body. For this reason, it was necessary to develop an effective method for the size distribution analysis of residual HCD fragments with high accuracy and sensitivity. In this study, capillary gel electrophoresis with laser-induced fluorescence detector (CGE-LIF) was used to analyze the size distribution of residual HCD fragments in lentivirus products. The results confirmed that lentiviral RNA genome could interfere with the size distribution analysis of residual HCD fragments. By optimizing the amount of RNase I and digestion time in sample pretreatment process, the interfere of RNA genome could be avoided. The specificity, precision, accuracy, linear range, the detection of limit (LOD), and the quantification of limit (LOQ) of CGE-LIF method were also validated. The results showed that the CGE-LIF method had a good performance both in terms of specificity and reproducibility. The intra-and inter-day relative standard deviations of migration time and corrected peak area were all less than 1% and 2%, respectively. The 200 bp DNA marker had a good linearity between 50 and 1000 pg/ml. The LOD and LOQ of 200 bp DNA marker were 2.59 and 8.64 pg/ml, respectively. In addition, this method was successfully used to analyze the size distribution analysis of residual HCD fragments in lentivirus products with different production processes.

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A platform method for plasmid isoforms analysis by capillary gel electrophoresis

Cited by 10 publications

References 17 publications

Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments

Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments

Gels in Microscale Electrophoresis

Size distribution analysis of residual host cell DNA fragments in lentivirus by CGE‐LIF

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