The Detection of Nucleic Acids Following Electrophoretic Separation
Robin Martin in Gel Electrophoresis: Nucleic Acids, 2020
No information can be obtained from an electrophoretic separation unless the positions of the nucleic acids can be detected and recorded. Whilst the focus is the theory and practice of nucleic acid electrophoresis, it would be a serious omission not to review the variety of methods that exist for detecting DNA and RNA molecules once an electrophoretic separation has been achieved. There are a number of dyes that possess the useful property of low fluorescence when free in solution, but high fluorescence when bound to DNA or RNA. The most widely used compound is ethidium bromide. Agarose or polyacrylamide gels that contain ethidium bromide should be collected for incineration with clinical waste. Techniques that employ labeling of DNA or RNA with fluorescent nucleotides use a fundamentally different principle to detect and record the separation of nucleic acids during gel electrophoresis.
Nucleotide Sequencing and Hybridizing
Karl Kammermeyer, Virginia L. Clark in Genetic Engineering Fundamentals, 2017
Nucleotide sequencing is the analytical chemistry of genetic engineering in general and recombinant techniques in particular. The procedure uses the well-known method of electrophoresis where organic molecules of different size will travel at different rates in a gel matrix under the influence of an electric potential (direct current); larger molecules move more slowly because of their interaction with the gel structure. Thus, DNA chains of varying nucleotide content (complexes of P-S-B) * which have been labeled by 32 P insertion at 5′ end will spread on the gel into discrete sequential position. The location of the respective nucleotides is visualized on the gel by staining with ethidium bromide (EtBr) or a fluorescent dye. The EtBr or dye fluoresce under ultraviolet light and a photographic record can be established. Also, when only small amounts of DNA are present, subsequent exposure to an x-ray film gives an autoradiograph of 32 P location which then can be read to ascertain the order of bases.
Exercise 6: Polymerase Chain Reaction (PCR)-Based Tests: The AmpliType PM/DQA1 System
J. Thomas McClintock in Forensic DNA Analysis, 2008
If the evidentiary sample contains an insufficient quantity of DNA or if the DNA is degraded, a polymerase chain reaction (PCR)–based test may be used to obtain a DNA profile. The PCR-based tests generally provide rapid results that can serve as an alternative or as a complement to other DNA testing. The process involves the isolation of DNA from a biological specimen (e.g., blood, semen, saliva, or fingernail clippings) followed by an assessment of DNA quality and quantity. Next, the PCR amplification technique is used to produce millions of copies of a specific portion of a targeted DNA segment (Figure 7). The PCR amplification procedure is similar to a molecular photocopying machine. The amplified PCR products are then separated and identified by the reverse dot blot technology or by gel electrophoresis followed by enzymatic conversion of a colorless substrate or by chemical staining using ethidium bromide or coomassie blue, respectively. The resulting DNA profiles are routinely interpreted by direct comparison to DNA and/or allele standards. Probability calculations are determined based upon classical population genetic principles.
Differentiation Effect of Pyruvate and Uridine on Cultured U937-ρ° Cells
Published in Ultrastructural Pathology, 2009
Yishan Liu, Li Geng, Zhenhe Suo
The human pro-monocytic leukemia U937 cell line was previously reported to become ρ°cells after a long-term ethidium bromide exposure. In the authors' extensive PCR studies with different pairs of primers for the mtDNA molecule they showed that these U937-ρ° cells, after being cultured in their laboratory for a time, did replete their mtDNA. That the cells grew well in the normal medium (RPMI 1640 plus 10% fetal calf serum and 2 mM L-glutamine) as the parental cells also suggests that these cells contain functional mitochondria and mtDNA molecules. Further experiments showed that the cells cultured in the medium with pyruvate and uridine rather rapidly and strongly adhered on the culture flask walls while the cells cultured in the medium without pyruvate and uridine either floated or only very loosely covered the culture flask walls. Ultrastructural examination showed that the floating cells, which were often found in the culture without pyruvate and uridine, were rather similar to monocytes in nature, like the original U937 cells, while the attached larger cells appearing earlier in the culture with pyruvate and uridine demonstrated macrophage differentiation, indicating a differentiation effect of pyruvate and uridine on the cells.
Naringenin; a bioflavonoid, impairs the reproductive potential of male mice
Published in Toxicology Mechanisms and Methods, 2017
Pavitra Ranawat, Nikita Bakshi
Present study analyzed the effect of naringenin, a bioflavonoid, on male reproductive function in adult mouse, after intraperitoneal treatment with varying concentrations of naringenin (2, 8 and 20 mg/kg b.wt.) for two weeks. Naringenin increased the generation of reactive oxygen species and lipid peroxidation in the testis with concomitant decrease in sperm count and motility in a dose-dependent manner. Activities of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase and levels of reduced glutathione were found to be decreased in a dose-dependent manner. Also, the levels of oxidized glutathione were increased leading to a shift in redox ratio. Naringenin treatment also led to a dose-dependent increase in the mRNA expression of c-jun, c-fos and NF-κB. The testicular histomorphology was also altered dose dependently. Additionally, the number of apoptotic germ cells increased with increasing doses of naringenin as evident from acridine orange/ethidium bromide costaining and JC-1 staining. In conclusion, our study reveals that naringenin despite being a potent antioxidant with numerous important biological functions may also act as pro-oxidant, thus causing damaging effects in the testicular tissue.
Preparation of polysulfone hollow microspheres encapsulating DNA and their functional utilization
Published in Journal of Microencapsulation, 2004
C. Zhao, X. D. Liu, M. Nomizu, N. Nishi
Polysulfone hollow microspheres encapsulating DNA were prepared using a liquid–liquid phase separation technique. The microspheres were then used to absorb a DNA-binding intercalating material — ethidium bromide. The amount of DNA encapsulated in the microspheres depended on the concentration of the DNA solution used to prepare the microspheres, and the microsphere morphology depended on both the polymer concentration and the preparation conditions. The amount of ethidium bromide in the microspheres depended mainly on the amount of encapsulated DNA, and the microsphere morphology also affected the removal of the ethidium bromide. The new method of DNA encapsulation is proposed, and the microspheres encapsulating the DNA have the potential to be used in environmental applications.
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