HLA-DR and -DQ Typing by DNA-RFLP Analysis
M. Kam, Jeffrey L. Bidwell in Handbook of HLA TYPING TECHNIQUES, 2020
RFLP analysis was first applied to the investigation of HLA class II polymorphism by Wake et al.30 The technique (Figure 1) involves hybridization of labeled, single-stranded DNA sequences (probes) to homologous single-stranded DNA sequences (targets) within genomic DNA. Prior to hybridization, genomic DNA is treated by digestion with a restriction endonuclease; endonucleolytic fragments are then separated according to size by agarose gel electrophoresis and are transferred to a solid support by capillary or vacuum blotting. Although originally advocated as a technique with only a supplementary role in tissue typing, subsequent studies have shown that RFLP analysis permits the accurate genotyping of most DR, DQ, and Dw specificities.1-4-11-12-23-28 These investigations have shown that the correlation between RFLP and phenotypically determined DR, DQ, and Dw specificities varies according to the choice of restriction endonuclease employed.
Cellular and Molecular Mechanisms of Ischemic Acute Renal Failure and Repair
Robin S. Goldstein in Mechanisms of Injury in Renal Disease and Toxicity, 2020
The pathological features of necrosis are very different from those of programmed cell death (Searle, et al., 1982). The latter has been called “apoptosis”, a term coined by Kerr et al., who attributes the derivation of the term to Professor James Cormack of the University of Aberdeen (Kerr, et al., 1972). “Apoptosis” is a Greek term to describe “dropping off’ or “falling off’ of petals from a flower or leaves from a tree. Pathologically, the early stages of apoptosis are characterized by disappearance of microvilli, chromatin condensation at the periphery of the nucleus, condensation of cytosolic components, breakdown of epithelial desmosomal attachments, followed by cell surface protuberances, and fragmentation of the nucleus. Ultimately there is blebbing off of cell surface protuberances containing condensed cytosol and occasional nuclear fragments, to generate spherical or ovoid “apoptotic bodies” (Wyllie, et al., 1980) which can be phagocytosed by macrophages or epithelial cells. Apoptosis histologically is often associated biochemically with cleavage of double-stranded DNA at the linker regions between nucleosomes, resulting in fragments of approximately 180 to 200 bp (Kerr and Harmon, 1991). On agarose gel electrophoresis there is a characteristic ladder pattern. The endonuclease responsible for this biochemical correlate of apoptosis has not been well characterized. A candidate enzyme is an endonuclease that is activated by calcium in thymocytes (McConkey, et al., 1988). Increases in cellular calcium concentration can induce apoptosis (Kizaki, et al., 1989).
Apoptosis: Cellular Signaling and Molecular Mechanisms
John J. Lemasters, Constance Oliver in Cell Biology of Trauma, 2020
Internucleosomal DNA cleavage is observed only in cells undergoing apoptosis. Cellular DNA is organized into nucleosomes which are composed of octomers consisting of two molecules each of histones H2A, H2B, H3, and H4 around which the chromatin is wrapped. The DNA between such structures is known as the internucleosomal or linker region and is associated with histone H1.11 DNA cleavage occurs within this region during apoptosis, yielding fragments of approximately 180 b.p. (the length of DNA around one nucleosome).10 This DNA ladder can easily be observed by agarose gel electrophoresis, as shown in Figure 2. Human T-lymphoblastic leukemia cells (CEM-C7) undergoing apoptosis have been reported to have double stranded fragments which are blunt ended and possess 5′ phosphate and 3′ hydroxyl groups, but whether apoptotic DNA cleavage products from other cell types or whole animals are similar is unclear since the cell death program in CEM-C7 cells is atypical.12 It remains uncertain whether DNA degradation is actually specific for particular genes, and whether susceptibility to nuclease digestion is dependent on DNA conformation. Studies in lens fibers undergoing differentiation and in hen oviduct nuclei suggest that certain genes are preferentially inactivated during apoptosis.13 Conversely, sequencing of randomly selected chromatin fragments from cells undergoing γ-radiation-induced apoptosis suggests that cleavage occurs preferentially in regions of DNA possessing non-B conformation and is not specific for particular genes.14
The induction of aryl hydrocarbon receptor (AHR) in immune organs of developing chicks by polycyclic aromatic hydrocarbons
Published in Toxicology Mechanisms and Methods, 2018
A. R. Nisha, H. Hazilawati, M. L. Mohd Azmi, M. M. Noordin
Agarose gel electrophoresis was done using 7 × 10 cm Mini Sub cell Gel electrophoresis apparatus (Bio-Rad, Hercules, CA) as described in a manual (Green and Sambrook 2012). The 1% agarose gel was prepared by adding 1 g of agarose powder in 100 mL TAE (tris acetate EDTA) and boiled until the powder is dissolved. The agarose solution was cooled down to about 60 °C before pouring into gel tray that has been placed in an eight welled comb and bordered with a gel casting gates within the gel electrophoresis apparatus. The gel was allowed to solidify at room temperature and gently the comb and gates were removed. TAE buffer was poured into the electrophoresis apparatus to submerge the solidified gel. DNA ladder of 100 bp was used as DNA marker (Promega, Madison, WI). The DNA ladder marker was prepared by mixing 10 µL markers and 2 µL of 6× loading dye. Other wells were loaded with 10 µL of RT PCR products that had been premixed with 2 µL of loading dye. Gel electrophoresis was run at 80 V for 35 minutes. After electrophoresis, the gel was transferred from the electrophoresis apparatus and submerged into ethidium bromide staining solution for 15–30 minutes. The gel was destained with distilled water for 15 minutes and placed in UV transilluminator to visualize the nucleic acid bands.
Loop-mediated isothermal amplification assay as a point-of-care diagnostic tool for Vibrio parahaemolyticus: recent developments and improvements
Published in Expert Review of Molecular Diagnostics, 2019
Karanth Padyana Anupama, Anirban Chakraborty, Iddya Karunasagar, Indrani Karunasagar, Biswajit Maiti
LAMP assay is a unique form of DNA amplification developed by Notomi et al. [27]. The assay involves simple pre and post-processing methods. From sample collection to completion of the test it requires very less time when compared to other molecular methods (Figure 1). Therefore, LAMP has the potential of a point-of-care diagnostic tool for identification of pathogens such as V. parahaemolyticus. One of the important pre-requisites in any molecular diagnostic technique is the use of purified DNA. It is generally time-consuming and cost-intensive to obtain pure DNA. However, in the LAMP, this pre-requisite can be done away with since crude DNA can be used as a template for the reaction. Moreover, the end products can be detected by multiple methods such as- a) using agarose gel electrophoresis b) visualization by the naked eye c) using a turbidimeter [53–55]. The disadvantages of the LAMP assay are very limited, the most crucial being the chances of contamination. Since the assay uses four sets of primers, it offers high specificity and can detect DNA at the femtogram level. This makes the assay highly susceptible to contamination. To avoid false positive results, adequate precaution should be taken while performing the assay. The advantages and disadvantages of the LAMP assay and its variants are listed in Table 2.
Cyclic peptide-based nanostructures as efficient siRNA carriers
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Bijayananda Panigrahi, Rohit Kumar Singh, Sourav Mishra, Dindyal Mandal
The binding assay was performed using 100 pmol siRNA and different concentrations of cyclic peptides ranging from 1:10 to 1:100 in 20 µl DMEM without serum and incubated for 30 min at room temperature. Then, samples were analyzed in agarose gel electrophoresis (Model: Bio-Rad, California, USA) using 2% agarose gel (w/v) impregnated with ethidium bromide for 30 min at 80 V. TAE buffer (5 mM Tris base, 2.5 mM acetic acids, 0.1 mM Na2EDTA) was used as an electrophoresis running buffer. Gels were scanned by Gel Doc™ EZ Bio-Rad imager. In order to investigate the stability of peptide–siRNA complex, the physical mixture was treated with 10% FBS, 1 N HCl,1N NaOH, 5 mM glutathione, 2 nmol proteinase K, 510 nmol trypsin, respectively, for 30 min. Effect of RNase A was investigated by incubating the mixture with RNase A for 5 min.
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