Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR

Yang, Inchul; Park, In Young; Jang, Sung-Moon; Shi, Lian Hua; Ku, Hyung-Keun; Park, Sang-Ryoul

doi:10.1093/nar/gkl257

Cited by 21 publications

(10 citation statements)

References 27 publications

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“…6,7 Recently, many approaches for quantitative assessment of DNA methylation depending on sodium bisulfite treatment of genomic DNA and subsequent PCR amplification have been developed and widely used. [8][9][10] In these methods, when DNA is treated with a high concentration of bisulfite, unmethylated cytosine is converted into uracil, whereas methylated cytosine remains intact. If a target gene is amplified by PCR, the PCR product will contain thymine at a general cytosine location and cytosine at a methyl cytosine location.…”

mentioning

confidence: 99%

Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces

Jiang

Zhang

Jing

et al. 2010

Laboratory Investigation

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Various technologies are currently available to quantify DNA methylation. However, rapid and simple methods for determining the DNA methylation status of CpG sites in genes still remain elusive. In this report, we describe a novel method for the rapid quantification of CpG methylation on the basis of direct bisulfite-PCR sequencing method. According to the principles of bisulfite-PCR, converting unmethylated cytosines to thymine while leaving methylated cytosines unchanged, we regard the CpG site as a SNP and estimate the methylation status of cytosines in the given CG dinucleotides by measuring the ratio of the cytosine peak height to the sum of cytosine and thymine peak heights in automated DNA sequencing traces. Furthermore, we take several effective measures to break through the 'bottleneck' problems that render the routine bisulfite sequencing method unsuitable for quantitative methylation. In comparison with pyrosequencing and bisulfite-cloning sequencing, our method is confirmed to be a simple, high-throughput and cost-effective technology for determining the methylation status of specific genes. Accordingly, this novel method is anticipated to be an efficient and economical alternative tool for rapid quantification of methylation patterns in screening large numbers of clinical samples across multiple genes.

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confidence: 99%

Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces

Jiang

Zhang

Jing

et al. 2010

Laboratory Investigation

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“…Some modifications to the method described in were necessary, starting from the injection mode: in electrokinetic injection, analytes are quantitatively injected in the capillary on the basis of their mobility. Due to lack of data in the literature about mobility of dCMP and d5mCMP, pressure mode injection was chosen in order to avoid errors when calculating the methylation ratio.…”

Section: Resultsmentioning

confidence: 99%

“…In this article, a novel method for detection of 2′‐deoxy‐5′‐methylcytosine 5′‐monophosphate (d5mCMP) was developed, starting from a previously published method for the analysis of 5′‐deoxynucleosides monophosphate (dNMP) in bisulfite treated PCR products . Full baseline resolution between 2′‐deoxycytosine 5′‐monophosphate (dCMP) and d5mCMP was obtained, through modification of the retention factor by increasing CTAB concentration and migration time window by the addition of glucose in the electrolyte.…”

Section: Introductionmentioning

confidence: 99%

Analysis of genomic methylation level using micellar electrokinetic chromatography with UV detection

et al. 2013

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Analytical methods for quantification of 5'-methylcytosine in genomes are important tools to investigate epigenetic changes in gene expression during development, differentiation, aging, or cancer. Here, we report a novel genomic methylation content assay based on enzymatic hydrolysis of DNA and MEKC separation of 5'-deoxyribonucleoside monophosphates (dNMP) using the cationic surfactant CTAB as pseudostationary phase. Calf Thymus DNA was used during method development to determine electrophoretic parameters and electrolyte composition for a complete separation between 2'-deoxycytosine-5'-monophosphate and 2'-deoxy-5'-methylcytosine 5'-monophosphate (d5mCMP). Methylated and not methylated oligonucleotides were used to confirm the identity of each peak and evaluate analytical parameters of the method. The LOD of the method was found to be 12.5 pmol/μL for d5mCMP.

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“…The degree of methylation was analyzed as a ‘C/T SNP’ using AQ mode of computer analysis software program (http://epigendx.com/d/service/pyrosequencing/global-dna-methylation/). Additionally, four CpG island sites were studied using PCR primers which represent the DNA methylation status for select genes with methylation grouped accordingly (high, medium, or low) relative to lean controls, utilizing previously established protocols with detailed explanation described elsewhere [33]. The degree (amount) of unknown original target sequence was then compared with the sequence after bisulfite conversion [34].…”

Section: Methodsmentioning

confidence: 99%

Examination of Global Methylation and Targeted Imprinted Genes in Prader- Willi Syndrome

Manzardo

2016

J Clin Epigenet

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Context Methylation changes observed in Prader-Willi syndrome (PWS) may impact global methylation as well as regional methylation status of imprinted genes on chromosome 15 (in cis) or other imprinted obesity-related genes on other chromosomes (in trans) leading to differential effects on gene expression impacting obesity phenotype unique to (PWS). Objective Characterize the global methylation profiles and methylation status for select imprinted genes associated with obesity phenotype in a well-characterized imprinted, obesity-related syndrome (PWS) relative to a cohort of obese and non-obese individuals. Design Global methylation was assayed using two methodologies: 1) enriched LINE-1 repeat sequences by EpigenDx and 2) ELISA-based immunoassay method sensitive to genomic 5-methylcytosine by Epigentek. Target gene methylation patterns at selected candidate obesity gene loci were determined using methylation-specific PCR. Setting Study participants were recruited as part of an ongoing research program on obesity-related genomics and Prader-Willi syndrome. Participants Individuals with non-syndromic obesity (N=26), leanness (N=26) and PWS (N=39). Results A detailed characterization of the imprinting status of select target genes within the critical PWS 15q11-q13 genomic region showed enhanced cis but not trans methylation of imprinted genes. No significant differences in global methylation were found between non-syndromic obese, PWS or non-obese controls. Intervention None. Main outcome measures Percentage methylation and the methylation index. Conclusion The methylation abnormality in PWS due to errors of genomic imprinting effects both upstream and downstream effectors in the 15q11-q13 region showing enhanced cis but not trans methylation of imprinted genes. Obesity in our subject cohorts did not appear to impact global methylation levels using the described methodology.

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Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR

Cited by 21 publications

References 27 publications

Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces

Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces

Analysis of genomic methylation level using micellar electrokinetic chromatography with UV detection

Examination of Global Methylation and Targeted Imprinted Genes in Prader- Willi Syndrome

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