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Detection Technology
Published in Rick Houghton, William Bennett, Emergency Characterization of Unknown Materials, 2020
Rick Houghton, William Bennett
Cooling the solution allows double-stranded DNA to remain intact. Subsequent heating and cooling cycles (thermocycling) will replicate the DNA exponentially, effectively amplifying the genetic sequence to an amount sufficient for detection (Figure 3.55). The increasing amount of target DNA can be detected in various ways. One technique uses ethidium bromide (EtBr), which will bind to double-stranded DNA and fluoresce. Over the course of thermocycling, the increase in fluorescence indicates an increasing amount of the target DNA. This method is effective even with samples that contain high amounts of nontargeted DNA because it detects an increase in fluorescence over the thermocycling periods. The fewer cycles necessary to produce a detectable increase in fluorescence, the greater the number of target sequences in the sample, which are duplicated exponentially by the PCR process. The cycle can occur as fast as every 17 seconds, and it is possible to get a billion-fold (32 thermocycles) in about 10 minutes, plus setup time.
Nano-Genotoxicity Evaluation: A Review
Published in Vineet Kumar, Nandita Dasgupta, Shivendu Ranjan, Nanotoxicology, 2018
Olusegun I. Ogunsuyi, Opeoluwa M. Fadoju, Motunrayo M. Coker, Solomon O. Akinrinade, Ifeoluwa T. Oyeyemi, Okunola A. Alabi, Chibuisi G. Alimba, Adekunle A. Bakare
The comet assay, also known as the single cell gel electrophoresis test, is the most frequently used genotoxicity assay in nanoparticle evaluation (Table 17.1). It is a simple, rapid, and sensitive test for quantification of low level DNA damages at the individual cell level. Comet assay detects single and double DNA strand breaks via migration of the damaged DNA in the gel electrophoresis (Tice et al. 2000). Comet assay was first used by Ostling and Johanson (1984) for quantification of DNA damages in irradiated murine lymphoma cell lines—L5178Y-S. Later, it was modified by Singh et al. (1988) into the popular comet assay version which can detect single and double strand breaks as well as alkali labile sites (Dhawan et al. 2009; Collins et al. 2014). It involves the suspension of a small number of cells in a thin layer of agarose gel on a microscope slide. The cells are lysed with detergent and treated with a high salt concentration. Subsequently, supercoiled DNA is released in the form of a nucleoid which unwinds. The slide is then passed to the electrophoresis chamber in order to separate the various strands obtained from the unwinding process according to molecular weights. The strands are subsequently stained with a fluorescent DNA-binding dye (e.g., ethidium bromide, propidium iodide). Cells with increased DNA damage migrate at a faster rate from the neucleoid toward the anode in the form of a comet. The amount of DNA migration is a quantifying indicator of DNA damage (Speit and Hartmann 2005).
Principles and Techniques for Deoxyribonucleic Acid (DNA) Manipulation
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
Nwadiuto (Diuto) Esiobu, Ifeoma M. Ezeonu, Francisca Nwaokorie
The 5’ phosphate groups of both DNA and RNA give them an overall negative charge which is exploited in nucleic acid extraction/isolation. When agarose is used as the matrix in gel electrophoresis, DNA and RNA molecules are separated on the basis of their molecular size and charge, as they migrate through the agarose gel pores. The pore size of the gel matrix is determined by the concentration of agarose; as concentration of agarose increases, the pore size will decrease. Therefore, larger molecules will move slower through the gel in comparison with the smaller DNA fragments. As electrical current passes through the gel, the DNA migrates moving from the cathode (−) to the anode (+); the smaller fragments moving faster (Figure 1.3b). Consequently, the smaller fragments migrate faster towards the positive anode. Other factors such as the nature of the DNA – supercoiled, relaxed, linear or circular – also affect their rate of migration through the gel matrix. Fluorescent dyes such as Sybr green and Ethidium bromide (EtBr) are used to visualize DNA bands under UV light. A gel-loading dye added to each sample permits the monitoring of the migration of DNA/RNA by running ahead since it contains an intensely colored dye (e.g. methylene blue) of low molecular weight. The loading dye also contains glycerol to increase the density of the DNA sample and allow it to fall within the wells/holes created within the agarose. Every gel run includes a reference of known molecular weight DNA markers, which helps assess the quality of the electrophoresis and is used to calculate the size of experimental DNA/RNA bands. Agarose gel electrophoresis is used to separate nucleic acid (DNA/RNA) ranging between 50 bp and ~15 kb. The migration rate of fragments is inversely proportional to the log of its molecular weight.
Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus correlates with disease severity in psoriasis: a case–control study
Published in Egyptian Journal of Basic and Applied Sciences, 2019
Doaa T. Masallat, Waleed Eldars, Ahmed F. State, Dalia Moemen
The clonal relationships between the MRSA isolates were studied by evaluating the genomic DNA with PFGE analysis that was performed according to the CDC protocol [20]. DNA was restricted with the SmaI enzyme and was separated on an agarose gel using a CHEF DR III apparatus (Bio-Rad laboratories). The running conditions were 6 V per cm, with pulses ranging from 5 to 40 s for 18 h at 14°C. The DNA banding patterns were visualized under UV light after staining with ethidium bromide (0.5 mg/mL). The similarities between the isolates were determined by visual comparison of the isolates band patterns. The interpretation of the PFGE results was carried out by eye according to the criteria described by Tenover et al. [21].
Spectroscopy, docking and molecular dynamics studies on the interaction between cis and trans palladium-alanine complexes with calf-thymus DNA and antitumor activities
Published in Journal of Coordination Chemistry, 2023
Asma Izadyar, Hassan Mansouri-Torshizi, Effat Dehghanian, Somaye Shahraki
Another method used to study the binding of coordinated complexes to DNA is the gel electrophoresis technique [36]. Different concentrations of the complexes (10, 15, 20 and 25 μL, from stock solutions) were prepared separately with 30 μl of DNA stock solution and the final volume of solutions was increased to 60 μl using Tris-HCl buffer was then incubated at 37 °C for 1 h. In the next step, the mixtures were mixed with DNA loading dye and then loaded onto the gel wells in TBE buffer and a constant voltage (90 V) was applied for 60 min. Note that at the time of preparation of the gel, 10 μL of ethidium bromide was added to visualize the DNA under UV light.
Evaluating the cytotoxicity of tin dioxide nanofibers
Published in Journal of Environmental Science and Health, Part A, 2018
Ashley S. Reynolds, Tanya H. Pierre, Rebecca McCall, Ji Wu, Worlanyo E. Gato
RNA concentration was measured using a NanoDrop 2000. Further, the quality of the RNA was determined using RNA gel electrophoresis stained with ethidium bromide. The required volume of RNA to be used in cDNA synthesis was determined. cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories, Hercules CA) by following their procedure.