Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications

Ziraldo, Riccardo; Shoura, Massa J.; Fire, Andrew; Levene, Stephen D.

doi:10.1093/nar/gkz534

“…Due to the compact distribution and overlapping of the larger fragment size peaks, the software groups several of these smaller peaks into one large peak. The software algorithms are also only capable of assigning accurate sizes and concentrations to the peaks of sufficiently resolved fragment populations, and attempt to do the same for large cluster peaks, thereby labelling the large peaks with sizes that are unrepresentative of the grouped sub-populations [ 36 ].…”

Section: Resultsmentioning

confidence: 99%

Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line

Ungerer

¹

,

Bronkhorst

²

,

Uhlig

³

et al. 2022

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Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed (hepta-nucleosomes were detectable with manual agarose gel electrophoresis), while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1–3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing.

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“…Introducing circularization presents a significant obstacle to extending quantitative electrophoresis methods, such as one proposed by Ziraldo et al (66). Calibrating gel electrophoresis measurements with a ladder of relaxed or nicked circular DNA of different lengths would be insufficient, since the contribution of the degree of supercoiling strongly affects the apparent DNA mass derived from the method of Ziraldo et al (at least by a factor of two).…”

Section: Discussionmentioning

confidence: 99%

DNA supercoiling-induced shapes alter minicircle hydrodynamic properties

Waszkiewicz

¹

,

Ranasinghe

²

,

Fogg

³

et al. 2023

Preprint

0

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DNA in cells is organized in negatively supercoiled loops. The resulting torsional and bending strain allows DNA to adopt a surprisingly wide variety of 3-D shapes. This interplay between negative supercoiling, looping, and shape influences how DNA is stored, replicated, transcribed, repaired, and likely every other aspect of DNA activity. To understand the consequences of negative supercoiling and curvature on the hydrodynamic properties of DNA, we submitted 336 bp and 672 bp DNA minicircles to analytical ultracentrifugation (AUC). We found that the diffusion coefficient, sedimentation coefficient, and the DNA hydrodynamic radius strongly depended on circularity, loop length, and degree of negative supercoiling. Because AUC cannot ascertain shape beyond degree of non-globularity, we applied linear elasticity theory to predict DNA shapes, and combined these with hydrodynamic calculations to interpret the AUC data, with reasonable agreement between theory and experiment. These complementary approaches, together with earlier electron cryotomography data, provide a framework for understanding and predicting the effects of supercoiling on the shape and hydrodynamic properties of DNA.

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“…Introducing circularization presents a significant obstacle to extending quantitative electrophoresis methods, such as one proposed by Ziraldo et al. ( 69 ). Calibrating gel electrophoresis measurements with a ladder of relaxed or nicked circular DNA of different lengths would be insufficient because the contribution of the degree of supercoiling strongly affects the apparent DNA mass derived from that method (at least by a factor of two).…”

Section: Discussionmentioning

confidence: 99%

DNA supercoiling-induced shapes alter minicircle hydrodynamic properties

Waszkiewicz

¹

,

Ranasinghe

²

,

Fogg

³

et al. 2023

Nucleic Acids Research

5

0

View full text Add to dashboard Cite

DNA in cells is organized in negatively supercoiled loops. The resulting torsional and bending strain allows DNA to adopt a surprisingly wide variety of 3-D shapes. This interplay between negative supercoiling, looping, and shape influences how DNA is stored, replicated, transcribed, repaired, and likely every other aspect of DNA activity. To understand the consequences of negative supercoiling and curvature on the hydrodynamic properties of DNA, we submitted 336 bp and 672 bp DNA minicircles to analytical ultracentrifugation (AUC). We found that the diffusion coefficient, sedimentation coefficient, and the DNA hydrodynamic radius strongly depended on circularity, loop length, and degree of negative supercoiling. Because AUC cannot ascertain shape beyond degree of non-globularity, we applied linear elasticity theory to predict DNA shapes, and combined these with hydrodynamic calculations to interpret the AUC data, with reasonable agreement between theory and experiment. These complementary approaches, together with earlier electron cryotomography data, provide a framework for understanding and predicting the effects of supercoiling on the shape and hydrodynamic properties of DNA.

show abstract

Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications

Cited by 12 publications

References 19 publications

Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line

Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line

DNA supercoiling-induced shapes alter minicircle hydrodynamic properties

DNA supercoiling-induced shapes alter minicircle hydrodynamic properties

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