scholarly journals Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR

2020 ◽  
Vol 41 (12) ◽  
pp. 2205-2216
Author(s):  
Rogier J. Nell ◽  
Debby Steenderen ◽  
Nino V. Menger ◽  
Thomas J. Weitering ◽  
Mieke Versluis ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Restriction Enzymes ◽  
Digital Pcr ◽  
Sodium Bisulfite ◽  
Bisulfite Conversion

scholarly journals Detection of LINE-1 hypomethylation in cfDNA of Esophageal Adenocarcinoma Patients

2020 ◽  
Vol 21 (4) ◽  
pp. 1547 ◽  
Author(s):  
Elisa Boldrin ◽  
Matteo Curtarello ◽  
Marco Dallan ◽  
Rita Alfieri ◽  
Stefano Realdon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Esophageal Adenocarcinoma ◽  
Methodological Approach ◽  
Methylation Status ◽  
Dna Amplification ◽  
Restriction Enzymes ◽  
Poor Quality ◽  
Bisulfite Conversion ◽  
Dna Hypomethylation ◽  
Methylation Patterns

DNA methylation plays an important role in cancer development. Cancer cells exhibit two types of DNA methylation alteration: site-specific hypermethylation at promoter of oncosuppressor genes and global DNA hypomethylation. This study evaluated the methylation patterns of long interspersed nuclear element (LINE-1) sequences which, due to their relative abundance in the genome, are considered a good surrogate indicator of global DNA methylation. LINE-1 methylation status was investigated in the cell-free DNA (cfDNA) of 21 patients, 19 with esophageal adenocarcinoma (EADC) and 2 with Barrett’s esophagus (BE). The two BE patients and one EADC patient were also analyzed longitudinally. Methylation status was analyzed using restriction enzymes and DNA amplification. This methodology was chosen to avoid bisulfite conversion, which we considered inadequate for cfDNA analysis. Indeed, cfDNA is characterized by poor quality and low concentration, and bisulfite conversion might worsen these conditions. Results showed that hypomethylated LINE-1 sequences are present in EADC cfDNA. Furthermore, longitudinal studies in BE suggested a correlation between methylation status of LINE-1 sequences in cfDNA and progression to EADC. In conclusion, our study indicated the feasibility of our methodological approach to detect hypomethylation events in cfDNA from EADC patients, and suggests LINE-1 methylation analysis as a new possible molecular assay to integrate into patient monitoring.

scholarly journals Quantification of DNA methylation using methylation-sensitive restriction enzymes and multiplex digital PCR

2019 ◽  
Author(s):  
R.J. Nell ◽  
D. van Steenderen ◽  
N.V. Menger ◽  
T.J. Weitering ◽  
M. Versluis ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Epigenetic Regulation ◽  
Dynamic Range ◽  
Restriction Enzymes ◽  
Digital Pcr ◽  
Tumour Suppressor Gene ◽  
Experimental Setup ◽  
Sodium Bisulphite ◽  
Allele Specificity ◽  
Reference Samples

ABSTRACTEpigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one well-studied aspect of epigenetic regulation, but is challenging to quantify accurately. In this study, we introduce a digital approach for the absolute quantification of the amount, density and allele-specificity of DNA methylation. Combining the efficiency of methylation-sensitive restriction enzymes with the quantitative power of digital PCR, DNA methylation is measured accurately without the need to treat the DNA samples with sodium bisulphite. Moreover, as the combination of PCR amplicon and restriction enzyme is flexible, the context and density of DNA methylation can be taken into account. Additionally, by extending the experimental setup to a multiplex digital PCR, methylation markers may be analysed together with physically linked genetic markers to determine the allele-specificity of the methylation. In-silico simulations demonstrated the mathematical validity of the experimental setup. Next the approach was validated in a variety of healthy and malignant reference samples in the context of RASSF1A promotor methylation. RASSF1A is an established tumour suppressor gene, that is aberrantly methylated in many human cancers. A dilution series of well-characterized reference samples cross-validated the sensitivity and dynamic range of the approach. Compared to conventional PCR based methods, digital PCR provides a more accurate and more sensitive approach to quantify DNA methylation. As no sodium bisulphite conversion is needed, also analysis of minute amounts of DNA could be carried out efficiently.

scholarly journals Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krystyna Ślaska-Kiss ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Pál Albert ◽  
Ákos Csábrádi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Binding Affinity ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Dna Binding Protein ◽  
E Coli ◽  
Dna Binding Affinity ◽  
Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

scholarly journals DNA Methylation in Solid Tumors: Functions and Methods of Detection

2021 ◽  
Vol 22 (8) ◽  
pp. 4247
Author(s):  
Andrea Martisova ◽  
Jitka Holcakova ◽  
Nasim Izadi ◽  
Ravery Sebuyoya ◽  
Roman Hrstka ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Solid Tumors ◽  
Epigenetic Modification ◽  
Single Gene ◽  
Restriction Enzymes ◽  
Methylation Analysis ◽  
Cpg Dinucleotides ◽  
Sequencing Technologies ◽  
Genome Level ◽  
Dna Methylation Analysis

DNA methylation, i.e., addition of methyl group to 5′-carbon of cytosine residues in CpG dinucleotides, is an important epigenetic modification regulating gene expression, and thus implied in many cellular processes. Deregulation of DNA methylation is strongly associated with onset of various diseases, including cancer. Here, we review how DNA methylation affects carcinogenesis process and give examples of solid tumors where aberrant DNA methylation is often present. We explain principles of methods developed for DNA methylation analysis at both single gene and whole genome level, based on (i) sodium bisulfite conversion, (ii) methylation-sensitive restriction enzymes, and (iii) interactions of 5-methylcytosine (5mC) with methyl-binding proteins or antibodies against 5mC. In addition to standard methods, we describe recent advances in next generation sequencing technologies applied to DNA methylation analysis, as well as in development of biosensors that represent their cheaper and faster alternatives. Most importantly, we highlight not only advantages, but also disadvantages and challenges of each method.

Cloning and Characterization of EphA3 (Hek) Gene Promoter: DNA Methylation Regulates Expression in Hematopoietic Tumor Cells

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2477-2486 ◽  
Author(s):  
Mirella Dottori ◽  
Michelle Down ◽  
Andreas Hüttmann ◽  
David R. Fitzpatrick ◽  
Andrew W. Boyd
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Expression Patterns ◽  
Gene Promoter ◽  
Restriction Enzymes ◽  
Clinical Samples ◽  
Cpg Dinucleotides ◽  
Normal Tissues

The Eph family of receptor tyrosine kinases (RTK) has restricted temporal and spatial expression patterns during development, and several members are also found to be upregulated in tumors. Very little is known of the promoter elements or regulatory factors required for expression of Eph RTK genes. In this report we describe the identification and characterization of the EphA3 gene promoter region. A region of 86 bp located at −348 bp to −262 bp upstream from the transcription start site was identified as the basal promoter. This region was shown to be active in both EphA3-expressing and -nonexpressing cell lines, contrasting with the widely different levels of EphA3 expression. We noted a region rich in CpG dinucleotides downstream of the basal promoter. Using Southern blot analyses with methylation-sensitive restriction enzymes and bisulfite sequencing of genomic DNA, sites of DNA methylation were identified in hematopoietic cell lines which correlated with their levels of EphA3 gene expression. We showed that EphA3 was not methylated in normal tissues but that a subset of clinical samples from leukemia patients showed extensive methylation, similar to that observed in cell lines. These results suggest that DNA methylation may be an important mechanism regulating EphA3 transcription in hematopoietic tumors.

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