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Role of Sperm DNA Damage in Male Infertility Assessment
Published in Botros Rizk, Ashok Agarwal, Edmund S. Sabanegh, Male Infertility in Reproductive Medicine, 2019
Saradha Baskaran, Chak-Lam Cho, Ashok Agarwal
The DBD-FISH quantifies DNA breaks and alkali-labile sites within a single cell [47,48]. In this technique, sperm cells are subjected to DNA denaturation and lysis to convert the DNA breaks into ssDNA and to remove membranes and proteins. The ssDNA is subsequently targeted for hybridization with fluorescent-labeled DNA probes. With a larger number of DNA breaks, the denaturation yields increased ssDNA, which hybridizes with DNA probes. The resulting intense FISH signal are captured and quantified using a digital image analysis system [49]. The main advantage of this technique is that it can be used to scan the whole-cellular DNA or specific DNA sequences of the sperm cells. The DBD-FISH is a reliable technique for determination of DNA breaks but the procedure is complex, expensive, and time consuming.
The Application of Genetic Tests in an Assisted Reproduction Unit: Sperm FISH
Published in Nicolás Garrido, Rocio Rivera, A Practical Guide to Sperm Analysis, 2017
Double-strand DNA denaturation of the sperm and FISH probes is carried out after incubation at high temperature (70°C–74°C). After denaturation, both DNAs are coincubated and hybridized to form a duplex of complementary strands. Hybridization protocols vary according to the type of FISH probe used, requiring different times and temperatures of hybridization (commonly between 4 and 16 hours at 37°C–42°C).
Cancer Detection and Prognosis
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Santiago Ropero, Manel Esteller
Bisulfite treatment converts all cytosine bases to uracil, except those that are methylated, are resistant to modification, and so remain as cytosines63 (Figure 14.2A). This reaction is the basis for differentiating methylated and unmethylated DNA. Bisulfite modification of DNA requires prior DNA denaturation because only methylcytosines located in single strands are susceptible to conversion. Total conversion of cytosine to uracil is a critical step in this process because unconverted unmethylated cytosine could be a source of false-positive results for methylated alleles. One disadvantage of bisulfite conversion is that these manipulations can lead to DNA fragmentation. This problem can be minimized by increasing the concentration of sodium bisulfite and reducing the time to complete the bisulfite reaction. Under these conditions, it is possible to obtain an optimal PCR product after only 4 hr of reaction.
Chromosome aberration in typical biological systems under exposure to low- and high-intensity magnetic fields
Published in Electromagnetic Biology and Medicine, 2020
Emanuele Calabrò, Hit Kishore Goswami, Salvatore Magazù
DNA denaturation is a physical-chemical process which has been largely studied in previous studies because it can provide a probe of the interactions that regulate physical-chemical mechanisms induced, for instance, by significant changes of temperature, pH or denaturant concentrations. During DNA denaturation its double helix structure unwinds separating into two single chains because of breaking of hydrogen bonds between (A-T) and (G-C) bases. Accurate description on thermal denaturation transition in B-DNA was obtained by neutron scattering (Wildes et al. 2011a, 2011b). The denaturation process of DNA can be induced also by physical-chemical stresses such as denaturant concentrations or exposure to ionizing radiations (Blake and Delcourt 1998; Borrego-Soto et al. 2015; Lomax et al. 2013; Reisz et al. 2014; Stellwagen et al. 2011; Tan and Chen 2006).
Genomics of Detoxification: How Genomics can be Used for Targeting Potential Intervention and Prevention Strategies Including Nutrition for Environmentally Acquired Illness
Published in Journal of the American College of Nutrition, 2020
Sharon R. Hausman-Cohen, Lee J. Hausman-Cohen, Grant E. Williams, Carol E. Bilich
Returning to toxicants, it is beneficial to look at one of the common endpoints of toxic damage to systems, Reactive Oxygen Species (ROS). Though this is not the only toxic endpoint this article will go into, it is certainly one of the most important. ROS, as the name implies, are molecules in the body that contain oxygen and are highly reactive. These ROS often function as oxidative agents, taking electrons from biological molecules such as lipids, DNA, and proteins. Created most prominently as natural byproducts of mitochondrial function, at healthy levels ROS serve in cell signaling to activate and deactivate various cellular pathways (3) and are functional parts of our immune system, utilized to harm pathogen invaders (4). Oxidation can also have a variety of deleterious effects, including the induction of mutation in DNA, denaturation of proteins resulting in a loss of function, or disruption of lipids and membranes that section off and protect cells (5). Thus, pathological increases in ROS levels contribute to a negative state described as oxidative stress. This can occur either due to increased generation or decreased clearance of reactive species—the latter, especially, is where genetic variants come into play.
Characterization of DNA cleavage produced by seminal plasma using leukocytes as a cell target
Published in Systems Biology in Reproductive Medicine, 2019
Elva I. Cortés-Gutiérrez, Carlos García De La Vega, Javier Bartolomé-Nebreda, Jaime Gosálvez
In our case, leukocytes processed with the halo-test show small halos of dispersed chromatin (baseline halos) emerging from the central nuclear core (Figure 2A). After applying the DBD-FISH protocol, these cells only exhibit positive but reduced hybridization in restricted areas of the core (Figure 2B). These regions indicate normal levels of susceptible DNA motifs to alkaline denaturation (ALS: alkali labile sites) which are prone to produce single strand DNA allowing DNA probe hybridization after DNA denaturation under mild alkaline conditions. Therefore, these labeled regions present at the core can be considered the constitutive hybridization signal for this cell type.