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Molecular Methods for Assessing Microbial Corrosion and Souring Potential in Oilfield Operations
Published in Kenneth Wunch, Marko Stipaničev, Max Frenzel, Microbial Bioinformatics in the Oil and Gas Industry, 2021
Gloria N. Okpala, Rita Eresia-Eke, Lisa M. Gieg
There are many DNA extraction procedures that are in use today for obtaining DNA suitable for sequencing. Steps involved in DNA extraction include cell lysis (to release nucleic acids from microbial cells), removal of inhibitors and cell debris that can interfere with downstream applications (e.g., PCR), and nucleic acid mobilization, purification, and elution into a suitable buffer (Figure 9.1) (Oldham et al. 2012). Cell lysis can be achieved by using either enzymes to disrupt cell wall and cell membrane components or chemicals or chaotropic agents that solubilize lipid membranes (Lever et al. 2015). However, total genomic DNA (gDNA) isolation using commercial kits that use a bead-beating step for cell lysis has been reported to give more consistent DNA yields (Cruaud et al. 2014; Yuan et al. 2015). Nonetheless, no commercial kit universally suits all samples. Overall, DNA extraction protocols may be modified or adjusted to improve DNA recovery from different samples (Morono et al. 2014).
Forensic DNA Profiling and Molecular Identification of Infectious Pathogens
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
D. E. Agbonlahor, M. Y. Tatfeng, Ifeoma B. Enweani-Nwokelo, Ifeoma M. Ezeonu, E. A. Brisibe, Francisca Nwaokorie, Nwadiuto (Diuto) Esiobu, A. O. Eremwanarue, F. E. Oviasogie, Benjamin Ewa Ubi, G. S. George
DNA Extraction: DNA extraction is the process by which scientists are able to separate the DNA from other macromolecules in a sample. It is a critical step molecular method, which provides information about the amount of DNA present in an unknown sample. This data can be used successfully to obtain better results preserving the sample for further analysis as shown in Figures 2.2 and 2.3, respectively.
Laboratory analyses of cyanobacteria and water chemistry
Published in Ingrid Chorus, Martin Welker, Toxic Cyanobacteria in Water, 2021
Judit Padisák, Ingrid Chorus, Martin Welker, Blahoslav Maršálek, Rainer Kurmayer
The conventional DNA extraction procedure uses a combination of osmotic shock and enzymatic treatment followed by chemical phase separation (e.g., Franche & Damerval, 1988). This DNA extraction procedure has been refined to obtain both qualitative and quantitative results, and today, robust protocols on cyanobacterial DNA extraction are available (e.g., Kurmayer et al., 2003). In some cases, extensive mucilage production as indicated by high viscosity of the DNA extract can inhibit the subsequent PCR amplification, and polysaccharides need to be selectively removed (Tillett & Neilan, 2000). In general, conventional DNA isolation procedure protocols are more time-consuming but cheaper than extraction with easy-to-use commercial DNA isolation kits which are widely available. Kit-based techniques typically include anion-exchange columns for DNA binding and purification. However, it is important to validate the efficiency of such techniques before using them for monitoring (Schober et al., 2007). DNA extraction from food supplements can be more difficult as for this purpose, cyanobacteria typically are processed by drying, and food supplements may contain additives that can affect DNA extraction efficiency, for example, pharmaceutical bulking agents with adsorbent properties (Costa et al., 2015). Thus, purification of DNA or alternatively the addition of substances reducing the effect of PCR inhibitors might be routinely required (Ramos et al., 2017a; b). Individual cyanobacterial colonies or filaments can be selected for PCR amplification of genes under a stereo microscope using a forceps or a micropipette (Kurmayer et al., 2002). Colonies or filaments are picked randomly from a subsample containing a few specimen only, washed by serial transfers in standard solution and stored in PCR buffer in the freezer (−20 °C). The DNA is extracted most efficiently by short sonification (Chen et al., 2016), and the obtained DNA quantities are sufficient for multiple individual PCR experiments.
Enhancement for the synthesis of bio-energy molecules (carbohydrates and lipids) in Desmodesmus subspicatus: experiments and optimization techniques
Published in Preparative Biochemistry & Biotechnology, 2023
Sreya Sarkar, Tridib Kumar Bhowmick, Kalyan Gayen
The DNA extraction process was employed to extract DNA from the cultured media and the DNA quality was assessed using 0.1% Agarose Gel. Afterwards 18S rDNA gene fragment was applied by NS1 and NS4 primers and then resolved on an Agarose gel. Subsequently purification of polymerase chain reaction (PCR) amplicon was performed to eliminate the contaminants. Following that, a forward and reverse DNA sequencing reaction of PCR amplicon was performed on ABI 3730xl Genetic Analyzer with forward and reverse primers using BDT v3.1 Cycle sequencing kit. Using aligner software, the 18S rDNA gene consensus sequence was produced by the help of forward and reverse sequence data. The first ten sequences were selected and aligned by applying a software programme (Clustal W) based on their maximum identity score. ClustalW is used to effectively align numerous nucleotide or protein sequences. It employs progressive alignment techniques that align the most identical sequences before progressing to the least similar sequences to produce a global alignment. Molecular evolution was analyzed using MEGA 7 to construct a phylogenetic tree and generate a distance matrix.
Removal of nonylphenol ethoxylate surfactant in batch reactors: emphasis on methanogenic potential and microbial community characterization under optimized conditions
Published in Environmental Technology, 2022
Rômulo Mota Teixeira, Isabel Kimiko Sakamoto, Fabrício Motteran, Franciele Pereira Camargo, Maria Bernadete Amâncio Varesche
Biomass samples from optimized anaerobic batch reactors (1.6 ± 0.10 mg.L−1 of NPE and 218.6 ± 10.6 mg.L−1 of ethanol) and control (without NPE) were collected at the end of the operation (after approximately 180 h) and washed with phosphate-buffered saline (PBS; 8% NaCL, 0.2% KCl, 1.44% Na2HPO4, 0.24%) KH2PO4 [47]. DNA extraction was performed using the phenolchloroform method (Sakamoto et al., 2019), and the extraction product was quantified (ng/µl) and purified (ratio 260/280 nm, rate ≥ 1.8) in an ND-2000 spectrophotometer (Thermo Fisher Scientific, USA), and DNA fragments were verified by agarose gel electrophoresis.