The Application of Genetic Tests in an Assisted Reproduction Unit: DNA Methylation Defects
Nicolás Garrido, Rocio Rivera in A Practical Guide to Sperm Analysis, 2017
Under certain conditions of pH and temperature, the sodium bisulfite converts the unmethylated cytosines (C) into uracil (U) by sulfonation, desulfonation, and deamination reactions (Figure 10.2). When the modified DNA is amplified by PCR, the C residues that are methylated are amplified as C and present a guanine (G) as a complementary base. On the contrary, the nonmethylated C turned to U are amplified as thymine (T) and presented an adenine (A) as complementary base. When analyzing the sequence of the PCR product, methylated/nonmethylated cytosine residues can be distinguished depending on the presence of C/G or A/T.
Analytical validation of a novel multi-target blood-based test to detect hepatocellular carcinoma
Published in Expert Review of Molecular Diagnostics, 2021
Andrea M. Johnson, Jeanne M. Dudek, David K. Edwards, Theran A. Myers, Patrick Joseph, Jennifer J. Laffin, Janelle J. Bruinsma
For the methylation marker workflow, DNA was extracted from 5–6 mL plasma or plasma surrogate using the QIAsymphony® SP instrument platform and reagents (Qiagen, Hilden, Germany). The DNA was transferred to the Hamilton® Microlab STARlet (Hamilton Robotics, Reno, NV) for bisulfite treatment, desulfonation, and subsequent re-purification using reagents manufactured by Exact Sciences Corporation (Madison, WI) [18]. To interrogate the methylation markers, 20 µL of the re-purified, bisulfite-converted DNA was amplified in one triplex assay – two HCC methylated DNA markers (MDMs), or regions of DNA that are hypermethylated in cancer cells compared to healthy cells, plus the constitutively methylated B3GALT6 reference marker – using the Long-probe Quantitative Amplified Signal (LQAS) chemistry. LQAS, which was run using the Quantstudio™ 5 Real-Time PCR Instrument (Life Technologies Corporation, Grand Island, NY) and reagents manufactured by Exact Sciences Corporation [18], combines amplification using real-time PCR and allele-specific detection of methylated target DNA through an invasive cleavage assay.
Childhood emotional neglect and oxytocin receptor variants: Association with limbic brain volumes
Published in The World Journal of Biological Psychiatry, 2020
Jacqueline Samantha Womersley, Sian Megan Joanna Hemmings, Christiane Ziegler, Ashley Gutridge, Fatima Ahmed-Leitao, David Rosenstein, Katharina Domschke, Soraya Seedat
DNA methylation was assessed according to the methods described by Ziegler et al. (2015). Briefly, 450 ng of DNA were treated with sodium bisulphite using the EZ DNA Methylation-Gold Kit (Zymo Research, HiSS Diagnostics GmbH, Freiburg, Germany) to convert non-methylated cytosines to uracil. Following desulfonation and purification, an amplicon covering parts of exon 3 of the OXTR gene, which includes 22 CpG sites, was amplified by PCR and the products purified and sequenced on the ABI 3730 XL platform. The resulting sequences were analysed using the Epigenetic Sequencing Methylation analysis software, as described previously (Domschke et al. 2012; Domschke et al. 2013; Domschke et al. 2014; Tadić et al. 2014; Domschke et al. 2015; Ziegler et al. 2016; Ziegler et al. 2017; Neyazi et al. 2018; Schiele et al. 2018). Due to technical difficulties in sequencing, especially at the 5’ and 3’ ends of the sequences, we obtained robust DNA methylation results for 12 of the 22 CpG sites, and thus have limited our analyses to this subset. Mean OXTR methylation data are available for all 63 of the study participants.
Single pot synthesized gold nanoparticles using Hippophae rhamnoides leaf and berry extract showed shape-dependent differential nanobiotechnological applications
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Further, 4-ABS may undergo desulfonation to form aniline (m/z 93) and sulfonic acid which are further mineralized to sulfurous acid (m/z 42) (Figure 9(D)). p-PDA undergoes deamination to form m-hydroxy benzoic acid ethyl ester (HBA, m/z 121) or aniline (m/z 93), which are transformed into short-chain carboxylic acids by oxidative ring opening, ultimately degrading to inorganic ions (sulfate, nitrate and ammonium), CO2 and H2O (Figure 9(D)). Figure 9(C) depicts structures of major identified products formed after the dye degradation.
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