Analysis of Epigenetic Modifications of DNA in Human Cells

Kristensen, Lasse Sommer; Treppendahl, Marianne Bach; Grønbæk, Kirsten

doi:10.1002/0471142905.hg2002s77

Cited by 12 publications

(11 citation statements)

References 101 publications

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“…Current needs in this field include ( i ) detailed experimental comparisons of results obtained with different PCR-based techniques (72), ( ii ) the availability of a large number of predesigned PCR-based DNA methylation assays to facilitate broad use, ( iii ) the implementation of minimum reporting guidelines for manuscripts describing results of PCR-based analyses of DNA methylation, including details of experimental conditions such as controls, primer sequences, and programs used for primer design (73), ( iv ) the further development of additional PCR-based techniques that allow DNA methylation measurements in a more quantitative and reproducible way (5), and ( v ) the implementation of automatic and multiplexed protocols for DNA methylation using currently available techniques to improve efficiency and reduce costs (59). For readers interested in genome-wide DNA methylation analysis, we recommend two available review articles (74,75).…”

Section: Discussionmentioning

confidence: 99%

“…The amount of DNA may be determined by spectrophotometric measurements using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA) (13) with settings for single-stranded DNA, or agarose gel electrophoresis and classical UV spectrometric analyses (5). Other more specific methods, such as qPCR (including MethyLight control assays) or PicoGreen may be more reliable and better suited for measuring limited amounts of DNA (14).…”

Section: Bisulfite Conversionmentioning

confidence: 99%

“…At present, the vast array of platforms available to study DNA methylation present a challenge for scientists who wish to enter this field (5). Among the methods for studying DNA methylation in candidate regions, PCR-based approaches have several advantages (6).…”

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confidence: 99%

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Optimizing methodologies for PCR-based DNA methylation analysis

et al. 2013

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Comprehensive analysis of DNA methylation patterns is critical for understanding the molecular basis of many human diseases. While hundreds of PCR-based DNA methylation studies are published every year, the selection and implementation of appropriate methods for these studies can be challenging for molecular genetics researchers not yet familiar with methylation analysis. Here we review the most commonly used PCR-based DNA methylation analysis techniques: bisulfite sequencing PCR (BSP), methylation specific PCR (MSP), MethyLight, and methylation-sensitive high resolution melting (MS-HRM). We provide critical analysis of the strengths and weaknesses of each approach as well as a series of guidelines to assist in selecting and implementing an appropriate method.

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Section: Discussionmentioning

confidence: 99%

Section: Bisulfite Conversionmentioning

confidence: 99%

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Optimizing methodologies for PCR-based DNA methylation analysis

et al. 2013

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“…This method requires four independent pyrosequencing reactions, one for each genotype and two negative controls using primers that do not recognize either allele and, in general, methods using MIP primers have a low analytical sensitivity compared to MSP based methods and may potentially be compromised by PCR bias2223.…”

Section: Discussionmentioning

confidence: 99%

Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing

Kristensen

Treppendahl

Asmar

et al. 2013

Sci Rep

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The tumor suppressor genes MGMT and DAPK1 become methylated in several cancers including diffuse large B-cell lymphoma (DLBCL). However, allelic methylation patterns have not been investigated in DLBCL. We developed a fast and cost-efficient method for the analysis of allelic methylation based on pyrosequencing of methylation specific PCR (MSP) products including a SNP. Allelic methylation patterns were reliably analyzed in standards of known allelic methylation status even when diluted in unmethylated DNA to below 1% methylation. When studying 148 DLBCL patients MGMT and DAPK1 methylation was observed in 19% and 89%, respectively, and among methylated and heterozygous patients 29% and 55%, respectively, were biallelically methylated. An association between the T-allele of the rs16906252 SNP and MGMT methylation was observed (p-value = 0.04), and DAPK1 methylation of the A-allele was associated with shorter overall survival (p-value = 0.006). In future cancer research allelic MSP-pyrosequencing may be used to study a wide range of other loci.

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“…Chemistry of DNA methylation and hydroxymethylation is summarized in Figure 1. DNA methyltransferases (DNMTs) catalyze the covalent addition of a methyl group from S-adenosyl-methionine (SAM) to the C5 position of cytosine in CpG dinucleotides (Figure 1), which predominantly cluster in densely populated CpG islands (CGI), often located in gene promoters and the first intron and exon [4–7]. Initially, 5-methylcytosine (5-mC) is established by de novo DNMT3A and DNMT3B [8], and during semi-conservative DNA replication, DNMT1 maintains methylation by using the hemi-methylated DNA template to regenerate the symmetrical methylation site on the new DNA strand [5].…”

Section: Introductionmentioning

confidence: 99%

Hydroxymethylation as a Novel Environmental Biosensor

Dao

Cheng

Revelo

et al. 2014

Curr Envir Health Rpt

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Beyond the genome, epigenetics has become a promising approach in understanding the interactions between the gene and the environment. Epigenetic regulation includes DNA methylation, histone modifications, and non-coding RNAs. Among these, DNA methylation, which is the addition of a methyl group to the fifth base of cytosine to produce 5-methylcytosine (5-mC), is most commonly studied. Epigenetic regulation has changed given the discovery of 5-hydroxymethylcytosine (5-hmC), considered the “sixth base”, and the nature of TET proteins to catalyze 5-mC oxidation to 5-hmC. 5-hydroxymethylation has been proposed to be a stable intermediate between methylation and demethylation and has raised questions about the functions of 5-hmC in gene regulation in cells, tissues, and organs in response to environmental exposure. Herein, we have provided an introduction to the chemistry of 5-hydroxymethylation, and the techniques for detection of 5-hydroxymethylation. In addition, we have reviewed current reports describing how 5-hmC responds to environmental factors, leading to the development of disease. And finally, we have discussed the potential use of 5-hmC in the study of disease development. All in all, it is our goal to provide innovative and convincing epigenetic studies for understanding the etiology of environmentally-related human disease, and translate these epigenetic findings into lifestyle recommendations and clinical practices to prevent and cure disease.

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Analysis of Epigenetic Modifications of DNA in Human Cells

Cited by 12 publications

References 101 publications

Optimizing methodologies for PCR-based DNA methylation analysis

Optimizing methodologies for PCR-based DNA methylation analysis

Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing

Hydroxymethylation as a Novel Environmental Biosensor

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