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Laboratory Molecular Methodologies to Analyze DNA Methylation
Published in Cristina Camprubí, Joan Blanco, Epigenetics and Assisted Reproduction, 2018
Restriction enzyme-based methods often utilize the advantage of parallel digestion of genomic DNA samples with enzyme isoschizomers. These enzymes cut at the same base position within the identical recognition sequences but differ in their sensitivities to methylation. The most commonly used isoschizomers for methylation studies are HpaII, which only cleaves unmethylated target sequences and MspI that cuts all recognition sites irrespective of the methylation status. Genomic DNA digested with methylation-sensitive restriction enzymes (MSRE) can be incorporated into a number of methods, some designed to estimate the levels at individual loci (quantitative qPCR and allelic PCR) or for low-resolution genome-wide methylation profiling such as MRE-seq (Table 5.1, Figure 5.1). During methylation-sensitive enzyme (MRE)-seq, the restriction enzyme cuts only the unmethylated positions resulting in DNA fragments that are small enough to generate size-selected libraries for NGS, thus revealing the location of unmethylated CpG sites.
Basic principles in cancer genetics
Published in J. K. Cowell, Molecular Genetics of Cancer, 2003
Before the advent of the large insert genomic clones, construction of long range physical genome maps was very difficult, and estimating distance between markers nearly impossible. This would change with the observation that certain restriction enzymes were sensitive to methylation at CpG dinucleotides. The best example is the pair of enzymes, MspI and HpaII. Both of these enzymes recognize the CCGG tetranucleotide and cleave DNA at this site. If the internal cytosine is methylated, however, HpaII will not cut the DNA. Thus, it was possible to analyze the methylation status of specific regions in the genome, and in particular the promotors of genes which are generally unmethylated, but where there was some suggestion that methylation could regulate gene expression. This analysis has proved very useful in the analysis of the P16 gene, for example, which frequently shows inactivation through promotor methylation. Misincorporation of thymidine at the site of a methyl-C during DNA replication is a common event which has resulted in the conversion for mammals into an A-T rich genome throughout evolution. Such changes only become important when they affect a critical nucleotide (resulting in a functional mutation if not repaired) or are involved in the regulation of genes. Since methylation occurs in CpGs, which are frequently seen in relatively high concentrations in the promotors of genes, there has been a strong selection pressure to maintain these regions of high GC. During early experiments with HpaII digestions of genomic DNA, Jack Miller noticed that a subpopulation of tiny fragments were generated in the 200–1000 bp range. These were called HpaII tiny fragments (HTF) and were shown to cluster at the beginnings of genes (Bird, 1986). In fact as other enzymes were identified which also had a high GC content in their recognition sequence, such as NotI, it became clear that the promotors of genes would also be restricted by these into small fragments compared with the rest of the genome, where these enzyme sites were ordinarily hundreds of kilobases apart. The frequent digestion by these so called ‘rare-cutter’ enzymes within the promotors of genes led to the description of HTF-islands in the genome. It has been estimated that up to 50% of all genes carry HTF islands in their promotors and this was often used to predict the approximate location of genes in large genomic regions and facilitate their cloning (Call et al., 1990).
Detection of a novel unbalanced X;21 translocation in a girl with Turner syndrome phenotype
Published in Gynecological Endocrinology, 2021
Elisavet Kouvidi, Sophia Zachaki, Nikoletta Selenti, Danai Veltra, Theodora Evmorfopoulou, Eirini Tsoutsou, Garifallia Tzifa, Christalena Sofocleous, Sarantis Gagos, Ariadni Mavrou
X inactivation studies were performed on genomic DNA extracted from peripheral blood using QIAcube automate extractor system (Qiagen GmbH, Hilden, Germany). The HUMARA assay (AR gene, locus: Xq12, CAG repeats) as well as methylation studies for PCSK1N (Xp11.23, CA/AG repeats) and FMR-1 (Xq27.3, CGG repeats) loci were performed. Digestion with HpaII and HhaI restriction enzymes, respectively, was applied and fluorescent PCR followed by capillary electrophoresis on ABI3500 analyzer was performed. The results were assessed with the Gene Marker software and allowed characterization of alleles and X-methylation profiling.
An integrative view on breast cancer signature panels
Published in Expert Review of Molecular Diagnostics, 2019
Zhen Wang, Xuanhao Zhang, Shuo Zhang, Xiaofeng Dai
Mammostrat™ uses the antibodies of five proteins to predict the recurrence risk of ER-positive, lymph node-negative patients treated with Tamoxifen, and stratify them into low-, moderate-, and high-risk groups [48]. These five proteins are TP53, N-myc downstream-regulated gene 1 (NDRG1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), solute carrier family 7 (cationic amino acid transporter, y+ system) member 5 (SLC7A5), and HpaII tiny fragments locus 9C (HTF9C). This assay is commercially available.
Epigenetic factors of individual radiosensitivity and adaptive capacity
Published in International Journal of Radiation Biology, 2020
Alexandra P. Kravets, Daryna A. Sokolova
Restriction analysis as well as amplification reactions were performed in a four-channel Tertsik DNA amplifier (DNA-Technology, Russia). Three types of restrictases were used: MspI (C…C*GG; C…CGG), HpaII (C…CGG) and MboI (…GATC*) (Fermentas, Germany). The restriction endonucleases HpaII and MspI both cleave the nucleotide sequence CCGG, but the action of HpaII is inhibited if the internal cytosine is methylated. MspI, an isoschizomer of HpaII which cleaves both unmethylated and methylated HpaII sites.