Electrophoretic Separation of DNA Fragments in Deuterated Water.
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| Year of publication | 2024 |
| Type | Conference abstract |
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| Description | Separation of DNA fragments according to their length is crucial for sequencing and other analyses. This is often performed via electrophoresis with fluorescence detection using various staining dyes. The intensity of the fluorescence signal can be affected by the physicochemical properties of the solvent (background electrolyte in case of electrophoresis), such as the temperature, pH, polarity, and other properties of the solvent. Compared to regular water, deuterated water provides lower fluorescence quenching and lower absorbance of the excitation radiation, which should result in a higher fluorescence signal [1]. This effect has been observed for various fluorophores. Deuterated water also affects the CE separation. It can decrease electroosmotic flow, thus improving the electrophoresis separation in electrolytes containing sieving matrices. This work aims to study the influence of heavy water on separation and detection limits for DNA electrophoresis on a chip and in a capillary. Selected DNA dyes were first characterized using a spectrofluorometer. Fluorescence intensities of the stained DNA in normal and deuterated water were compared to choose the most affected dyes for further electrophoretic experiments. The Agilent 2100 Bioanalyzer (red excitation) and 5200 Fragment Analyzer (blue excitation) were used for separations. The separation poly(n,n-dimethyl acrylamide) gels containing background electrolytes were prepared for both instruments in light and heavy water. For comparison, the gels from commercial kits were dried using vacuum and lyophilization, redissolved in normal and deuterated water, and used to separate DNA ladders labeled with selected fluorescent dyes on both instruments. An undocumented increase in the fluorescence signal of selected DNA-dye complexes was observed in several cases. Some experiments also confirm previously published [2] observations about longer separation times and higher resolution in heavy water-based solutions. |
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