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Keywords = Qubit fluorometry

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8 pages, 1187 KiB  
Commentary
Methodological Considerations Regarding the Quantification of DNA Impurities in the COVID-19 mRNA Vaccine Comirnaty®
by Brigitte König and Jürgen O. Kirchner
Methods Protoc. 2024, 7(3), 41; https://doi.org/10.3390/mps7030041 - 8 May 2024
Cited by 11 | Viewed by 65445
Abstract
DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a specific limit value must be set for them during marketing authorisation. This particularly applies to mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore, when quantifying [...] Read more.
DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a specific limit value must be set for them during marketing authorisation. This particularly applies to mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore, when quantifying the total DNA content in the final product, we must observe that, in addition to the mRNA active ingredient, DNA impurities are also encased in lipid nanoparticles and are therefore difficult to quantify. In fact, the manufacturer of the mRNA vaccine Comirnaty (BioNTech/Pfizer) only measures DNA impurities in the active substance by means of a quantitative polymerase chain reaction (qPCR), whose DNA target sequence is less than just 1% of the originally added DNA template. This means that no direct DNA quantification takes place, and compliance with the limit value for DNA contamination is only estimated from the qPCR data using mathematical extrapolation methods. However, it is also possible to dissolve the lipid nanoparticles with a detergent to directly measure DNA contamination in the final product by using fluorescence spectroscopic methods. Experimental testing of this approach confirms that reliable values can be obtained in this way. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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18 pages, 2671 KiB  
Article
The Influence of Tanning Chemical Agents on DNA Degradation: A Robust Procedure for the Analysis of Tanned Animal Hide—A Pilot Study
by Kristyna Hebenstreitova, Ondrej Salaba, Jakub Trubac, Jitka Kufnerova and Daniel Vanek
Life 2024, 14(1), 147; https://doi.org/10.3390/life14010147 - 19 Jan 2024
Cited by 4 | Viewed by 1805
Abstract
Illegal wildlife trade is currently on the rise, and it is becoming one of the most lucrative crime sectors. The rarer the species, the higher the demand. Wildlife trade falls under international regulations, such as the CITES convention. Proving that this convention has [...] Read more.
Illegal wildlife trade is currently on the rise, and it is becoming one of the most lucrative crime sectors. The rarer the species, the higher the demand. Wildlife trade falls under international regulations, such as the CITES convention. Proving that this convention has been violated is a complex process and can be very difficult to do. DNA analysis methods remain (in many cases) the only way to determine whether a certain specimen originated from a protected animal species, a specific individual, or a species in which it is legal to trade. Tanned animal hides are a specific type of specimen. With this type of biological material, obtaining amplifiable DNA is often difficult. This pilot study aimed to map the effect of the chemicals used in the tanning process on the degradation of the DNA yielded from such samples. The DNA was quantified using two different approaches: qPCR and Qubit fluorometry. The degree of DNA fragmentation was assessed by determining the degradation index. The results indicate that reagents containing chromium have the greatest influence on DNA degradation. However, by using the presented protocol, enough amplifiable DNA can be obtained from hides treated with aluminum-based reagents. Full article
(This article belongs to the Special Issue New Progress in Forensic Analysis of Animal and Plant Samples)
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14 pages, 798 KiB  
Article
Performance of Spectrophotometric and Fluorometric DNA Quantification Methods
by Brigitte Bruijns, Tina Hoekema, Lisa Oomens, Roald Tiggelaar and Han Gardeniers
Analytica 2022, 3(3), 371-384; https://doi.org/10.3390/analytica3030025 - 16 Sep 2022
Cited by 13 | Viewed by 16572
Abstract
Accurate DNA quantification is a highly important method within molecular biology. Methods widely used to quantify DNA are UV spectrometry and fluorometry. In this research, seven different DNA samples and one blank (MilliQ ultrapure water) were quantified by three analysts using one spectrophotometric [...] Read more.
Accurate DNA quantification is a highly important method within molecular biology. Methods widely used to quantify DNA are UV spectrometry and fluorometry. In this research, seven different DNA samples and one blank (MilliQ ultrapure water) were quantified by three analysts using one spectrophotometric (i.e., a NanoDrop instrument) and three fluorometric (i.e., the AccuGreen High Sensitivity kit, the AccuClear Ultra High Sensitivity kit, and the Qubit dsDNA HS Assay kit) methods. An analysis of variance (ANOVA) scheme was used to determine the influence of the analyst, the method, and the combination of analyst and method, on DNA quantification. For most samples, the measured DNA concentration was close to or slightly above the concentration of 10 ng/μL as specified by the supplier. Results obtained by the three analysts were equal. However, it was found that, compared to the fluorometric kits, the used spectrophotometric instrument in the case of fish DNA samples tends to overestimate the DNA concentration. Therefore, if sufficient sample volume is available, a combination of a spectrophotometric and a fluorometric method is recommended for obtaining data on the purity and the dsDNA concentration of a sample. Full article
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11 pages, 1878 KiB  
Protocol
Improved Method for DNA Extraction and Purification from Tetrahymena pyriformis
by Ezzouhra El Maaiden, Youssef El Kharrassi, Abdel Khalid Essamadi, Khadija Moustaid and Boubker Nasser
Methods Protoc. 2019, 2(2), 40; https://doi.org/10.3390/mps2020040 - 15 May 2019
Cited by 3 | Viewed by 9402
Abstract
Tetrahymena pyriformis (protozoa) is intensely investigated as a model organism, offering numerous advantages in comprehensive and multidisciplinary studies using morphologic or molecular methods. Since DNA extraction is a vital step of any molecular experiment, here a new mixed surfactant (Sodium dodecyl sulfate (SDS) [...] Read more.
Tetrahymena pyriformis (protozoa) is intensely investigated as a model organism, offering numerous advantages in comprehensive and multidisciplinary studies using morphologic or molecular methods. Since DNA extraction is a vital step of any molecular experiment, here a new mixed surfactant (Sodium dodecyl sulfate (SDS) 20%/Triton X-100) was adopted for effective DNA extraction from Tetrahymena pyriformis under an easy, fast protocol. The efficiency of this technique was then compared with three widely-used alternative techniques, namely the Chelex 100 matrix, Ammonium pyrrolidine dithiocarbamate (APD) complex and SDS–chloroform methods. DNA extraction was analyzed by pulsed-field gel electrophoresis, spectral measurement, fluorometry (Qubit), restriction enzyme digestion, and polymerase chain reaction. Data analysis revealed that the quantity and quality of the recovered DNA varied depending on the applied DNA extraction method. The new method (SDS 20%/Triton X-100) was the most efficient for extracting DNA from Tetrahymena pyriformis with high integrity and purity, affordable cost, less time, and suitability for molecular applications. Full article
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