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Enzymes Used for Recombinant DMA Technology Produced by Recombinant Microbes
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
The Taq DNA polymerase is a thermostable DNA polymerase originally purified from the extreme thermophile Thermus aquaticus [41]. This enzyme has a high optimum temperature and thermostability, which makes it an ideal enzyme for the use in PCR [42]. It also has a high processivity and capability to use deoxyribonucleotide triphosphate analogues, which makes it an excellent enzyme for DNA sequencing.
Carbohydrates and Nucleic Acids
Published in Michael B. Smith, A Q&A Approach to Organic Chemistry, 2020
A deoxyribonucleotide is a nucleic acid that uses 2-deoxyribose as the carbohydrate portion of the nucleotide. What is base pairing?
Effect of hubble-bubble smoking on global DNA methylation and transcription levels of protamine and histone genes in human spermatozoa
Published in Journal of Environmental Science and Health, Part A, 2023
Mohammed M. Laqqan, Maged M. Yassin
The terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to assess the percentage of spermatozoa DNA fragmentation.[7] This assay was conducted by using the in situ cell death detection kit according to the guidelines of the manufacturer (Roche Diagnostics, Germany). Briefly, sperm smears were prepared using ten microliters of semen suspension on slides and allowed to air-dry and then fixed with 4 percent paraformaldehyde phosphate-buffered saline, pH 7.4 at 22 degrees Celsius for twenty minutes, then rinsed with PBS. Sperm smears were then permeabilized with 0.1% Triton X-100 in 0.1 percent sodium citrate, pH 6.0 at 22 degrees Celsius for a third of an hour. Fifty microliters of the TdT-labeled nucleotide mixture (fifty microliters of enzyme solution and 450 microliters of label solution) were added to each sperm smear and incubated in a humidified chamber at 37 degrees Celsius overnight in a dark place. Negative controls without the TdT enzyme were run in each replicate. Then, the slides were rinsed three times in PBS and left to dry in the air followed by adding 25 microliters of five micrograms per millimeter DAPI stain solution to each slide as a counterstain and then covered by coverslips. For evaluation, 150 spermatozoa were analyzed on each slide by using the Zeiss Photomicroscope III. Finally, DNA fragmentation was estimated by distinguishing spermatozoa stained bright green “fragmented DNA, positive” from those stained dull green”(undamaged DNA, negative”.[32]
Assays and enumeration of bioaerosols-traditional approaches to modern practices
Published in Aerosol Science and Technology, 2020
Maria D. King, Ronald E. Lacey, Hyoungmook Pak, Andrew Fearing, Gabriela Ramos, Tatiana Baig, Brooke Smith, Alexandra Koustova
DNA sample preparation used for PCR amplification should be optimized for the successful amplification of increasingly longer targets from genomic DNA (Cheng et al. 1995). Therefore, before the PCR process, the intactness of the genomic DNA should be maintained during the collection and isolation process to create reproducible amplification of fragments with sizes >1.3 kb (Deagle, Eveson, and Jarman 2006). A larger target size can increase the probability of producing an unusable DNA template strand by randomly introducing a single-stranded (ss) nick or double-stranded (ds) break within the target sequence (Zhou, Pape, and Schwartz 2008). The DNA sample preparation varies for each experiment. Genomic DNA is isolated from the sample’s cultured cells by various methods such as boiling in the presence of a chelating resin, using alkaline lysis, or phenol extraction (Cheng et al. 1995). A standard reaction mixture contains the DNA template along with the specified forward and reverse primers, deoxyribonucleotide triphosphate (dNTP) mix, buffer with Mg++ for the specific DNA polymerase, and DNA polymerase. The PCR process typically utilizes thermal cycling, which consists of an initial denaturing step and a series of 20 to 40 cycles of denaturing, annealing and extension steps (Svabenska 2012; Liang and Johnson 1988; Sambrook, Fritsch, and Maniatis 1989).