Abstract 166: Absolute quantification of DNA methylation markers using methylation-sensitive restriction enzymes and multiplex digital PCR

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.

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

Human Mutation ◽

10.1002/humu.24111 ◽

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

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Absolute quantification of DNA methylation using microfluidic chip-based digital PCR

Biosensors and Bioelectronics ◽

10.1016/j.bios.2017.05.021 ◽

2017 ◽

Vol 96 ◽

pp. 339-344 ◽

Cited By ~ 26

Author(s):

Zhenhua Wu ◽

Yanan Bai ◽

Zule Cheng ◽

Fangming Liu ◽

Ping Wang ◽

...

Keyword(s):

Dna Methylation ◽

Microfluidic Chip ◽

Digital Pcr ◽

Absolute Quantification

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Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

Scientific Reports ◽

10.1038/s41598-021-83318-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tatsuhiko Hoshino ◽

Ryohei Nakao ◽

Hideyuki Doi ◽

Toshifumi Minamoto

Keyword(s):

Fish Species ◽

High Throughput Sequencing ◽

Correlation Coefficients ◽

Environmental Dna ◽

Digital Pcr ◽

Absolute Quantification ◽

Taqman Probe ◽

Individual Species ◽

Natural Environments ◽

Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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An integrated droplet digital PCR gene chip for absolute quantification of nucleic acid

Microfluidics and Nanofluidics ◽

10.1007/s10404-021-02465-4 ◽

2021 ◽

Vol 25 (7) ◽

Author(s):

Xiangkai Meng ◽

Yuanhua Yu ◽

Ping Gong ◽

Guangyong Jin

Keyword(s):

Nucleic Acid ◽

Digital Pcr ◽

Absolute Quantification ◽

Gene Chip ◽

Droplet Digital Pcr

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

Scientific Reports ◽

10.1038/s41598-021-94528-3 ◽

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.

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DNA Methylation in Solid Tumors: Functions and Methods of Detection

International Journal of Molecular Sciences ◽

10.3390/ijms22084247 ◽

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.

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Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors

Biological Procedures Online ◽

10.1186/s12575-021-00144-w ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Kevin Kuhlmann ◽

Melanie Cieselski ◽

Julia Schumann

Keyword(s):

Dynamic Range ◽

Genetic Material ◽

Housekeeping Genes ◽

Digital Pcr ◽

Absolute Quantification ◽

Test Procedures ◽

Experimental Conditions ◽

Rna Quantification ◽

The Stability ◽

Inflammatory Milieu

Abstract Background In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. Results There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. Conclusions To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

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Sepsis Diagnostics: Intensive Care Scoring Systems Superior to MicroRNA Biomarker Testing

Diagnostics ◽

10.3390/diagnostics10090701 ◽

2020 ◽

Vol 10 (9) ◽

pp. 701

Author(s):

Fabian Link ◽

Knut Krohn ◽

Anna-Maria Burgdorff ◽

Annett Christel ◽

Julia Schumann

Keyword(s):

Intensive Care ◽

Scoring Systems ◽

Digital Pcr ◽

Absolute Quantification ◽

Medical Problem ◽

Control Group ◽

Multiple Parameters ◽

Sepsis Diagnosis ◽

Intensive Care Patients ◽

Healthy Control

Sepsis represents a serious medical problem accounting for numerous deaths of critically ill patients in intensive care units (ICUs). An early, sensitive, and specific diagnosis is considered a key element for improving the outcome of sepsis patients. In addition to classical laboratory markers, ICU scoring systems and serum miRNAs are discussed as potential sepsis biomarkers. In the present prospective observational study, the suitability of miRNAs in sepsis diagnosis was tested based on proper validated and normalized data (i.e., absolute quantification by means of Droplet Digital PCR (ddPCR)) in direct comparison to classical sepsis markers and ICU scores within the same patient cohort. Therefore, blood samples of septic intensive care patients (n = 12) taken at day of admission at ICU were compared to non-septic intensive care patients (n = 12) and a healthy control group (n = 12). Our analysis indicates that all tested biomarkers have only a moderate informative power and do not allow an unequivocal differentiation between septic and non-septic ICU patients. In conclusion, there is no standalone laboratory parameter that enables a reliable diagnosis of sepsis. miRNAs are not superior to classical parameters in this respect. It seems recommendable to measure multiple parameters and scores and to interpret them with regard to the clinical presentation.

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