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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
While genomic (gDNA) and mitochondrial DNA (mtDNA) are present in nearly all the body’s cells (an important exception being red blood cells), DNA collection for the purposes of research is typically limited to buccal epithelial cells (cheek cells) or leukocytes (white blood cells). For studies in which blood drawing is a typical procedure, white cells (or the ‘buffy coat’) from a typical non-coagulated blood sample may be used for DNA isolation. If blood sampling is not required for a research study, then collection of buccal cells from the inside of the cheek using a cotton-type swab is a less invasive way to collect DNA. These cells are then processed to burst (‘lyse’) the cells and release the DNA from the cell nuclei, most often using commercially available DNA isolation kits (e.g. DNeasy Blood and Tissue Kit, Qiagen, Manchester, UK), which contain the chemicals needed for cell lysis, protein denaturation (to remove histone and other proteins from the DNA) and purify the remaining DNA. DNA isolation typically requires a couple of hours using the reagents provided in the kits across multiple steps involving centrifugation at high speeds, but many samples can be processed at the same time for efficiency. Once DNA is collected and purified, it can be stored in a sealed tube in a typical refrigerator or freezer for many years; it is one of the most stable biological molecules known. This DNA can then be assessed across a variety of DNA analysis methods typically employed in the field of molecular exercise physiology.
The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Measurements of genetic variability can be used to establish a link between the variations and disease, and to predict a patient’s therapeutic response to a drug along with potential toxicological outcomes. After completion of the Human Genome Project (HGP) between 2000 and 2003, the next important step was the discovery of new nonredundant genetic markers known as Single-Nucleotide Polymorphisms or “SNPs”. This involved DNA isolation followed by nucleic acid amplification and product detection. Originally, this was a laborious process involving Southern Blots and manual di-deoxy sequencing. The first technological revolution was based on the polymerase chain reaction (PCR), and included assays such as PCR Restriction Length Fragment Polymorphism (PCR-RLFP), PCR-ligase Detection, PCR Fluorescent Resonance Energy Transfer (PCR-FRET), and the nonamplified FRET assay (Invader Assay). The next improvement resulted from automated sequencing instruments (i.e., DNA Sequencers), which allowed high-throughput nucleotide sequencing and pyrosequencing. Finally, a revolution in SNP analysis resulted from the development of SNP Arrays and accompanying bioinformatics software that enabled large-scale linkages to be identified along with association and copy number studies in a low-cost, high-throughput fashion. Based on these developments, a large range of commercial instruments is now available that allow simultaneous measurement of thousands of SNPs across numerous samples using standard protocols, reagents, and data analysis systems.
Synthesis of Important Chiral Building Blocks for Pharmaceuticals Using Lactobacillus and Rhodococcus Alcohol Dehydrogenases
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Marion Rauter, Simon Krebs, Gotthard Kunze
Screenings from metagenomes allow DNA isolation from an environmental sample without microorganism cultivation. Itoh et al. (2014) identified two candidate genes out of 240 putative ADHs from a soil sample for the synthesis of chiral alcohols such as ethyl (R)-3-hydroxybutanoate.
Genetic alterations of C9orf72, SOD1, TARDBP, FUS, and UBQLN2 genes in patients with Amyotrophic Lateral Sclerosis
Published in Cogent Medicine, 2019
Ciftci Vildan, Darbas Sule, Bilgen Turker, Uysal Hilmi, Karauzum Berker Sibel
The study was supported by the ethics committee of Akdeniz University Faculty of Medicine Clinical Research. The study included a total of 30 patients diagnosed with ALS at Akdeniz University Faculty of Medicine Department of Neurology. Among those, 12 had fALS from six different families and 18 had sALS. Genomic DNA isolation was performed from the peripheral blood samples by using salting out method (Miller, Dykes, & Polesky, 1988). The hexanucleotide repeat region of the first intron of C9orf72 gene was amplified by the triplet primed PCR method and analyzed via fragment analysis. Five exons of the SOD1 gene, including exon–intron junction sites, were amplified using five different primer pairs. FUS gene exons; 3, 4, 5, 6, 11, 12, 13, 14, and 15 which are carrying the most common mutations, and the exon–intron junction sites were amplified using six different primer pairs. All sequences coding the TARDBP gene and the exon–intron junction sites were amplified using five different primer pairs, UBQLN2 gene that has no introns were amplified using four different primer pairs.
Inhalation of PM2.5 from diesel exhaust promote impairment of mitochondrial bioenergetics and dysregulate mitochondrial quality in rat heart: implications in isoproterenol-induced myocardial infarction model
Published in Inhalation Toxicology, 2022
Bhavana Sivakumar, Gino A. Kurian
The DNA isolation was done using phenol-chloroform-isoamyl alcohol method according to manufacturer’s instructions. The concentration of total DNA was measured spectrophotometrically using a Nanodrop ND-1000 spectrophotometer. The mRNA extraction was carried out using TRIzol reagent (15596026, Thermo Scientific, Waltham, MA) as previously described (Shanmugam et al. 2018). The expression of mitochondrial fission, fusion, mitophagy genes, and mitochondrial copy number were measured in triplicate, and qPCR results obtained were confirmed by three independent experiments. PCR was performed on Step-One Plus PCR system from Applied Biosystems (Waltham, MA). For data analysis, the ΔΔCt method was used to analyze differences in fold-changes in gene expression.
Polystyrene nanoparticles: the mechanism of their genotoxicity in human peripheral blood mononuclear cells
Published in Nanotoxicology, 2022
Kinga Malinowska, Bożena Bukowska, Ireneusz Piwoński, Marek Foksiński, Aneta Kisielewska, Ewelina Zarakowska, Daniel Gackowski, Paulina Sicińska
The most important DNA oxidation biomarkers are oxidized products of guanine and deoxyguanosine, which include 8-oxo-2′-deoxyguanosine (8-oxodG). To detect 8-oxodG, PBMCs were exposed to PS-NPs in concentration range from 0.0001 to 100 µg/mL for 24 h. The cells were subsequently frozen. In the next stage, DNA isolation was performed, as described in the Section 2.1.2, and the tested parameter was determined in the obtained DNA isolates. Two-dimensional (2D) liquid chromatography was used for the 8-oxodG analysis. Changes in the tested parameter were observed only after PBMCs exposure to the smallest size NPs. A statistically significant increase in 8-oxodG occurred from their concentration of 0.1 µg/mL (Figure 10).