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Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR
Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA m...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756443/ https://www.ncbi.nlm.nih.gov/pubmed/32906203 http://dx.doi.org/10.1002/humu.24111 |
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author | Nell, Rogier J. van Steenderen, Debby Menger, Nino V. Weitering, Thomas J. Versluis, Mieke van der Velden, Pieter A. |
author_facet | Nell, Rogier J. van Steenderen, Debby Menger, Nino V. Weitering, Thomas J. Versluis, Mieke van der Velden, Pieter A. |
author_sort | Nell, Rogier J. |
collection | PubMed |
description | Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation‐sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context‐density of methylation can also be determined, biological mechanisms can now be quantitatively assessed. |
format | Online Article Text |
id | pubmed-7756443 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-77564432020-12-28 Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR Nell, Rogier J. van Steenderen, Debby Menger, Nino V. Weitering, Thomas J. Versluis, Mieke van der Velden, Pieter A. Hum Mutat Methods Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation‐sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context‐density of methylation can also be determined, biological mechanisms can now be quantitatively assessed. John Wiley and Sons Inc. 2020-09-19 2020-12 /pmc/articles/PMC7756443/ /pubmed/32906203 http://dx.doi.org/10.1002/humu.24111 Text en © 2020 The Authors. Human Mutation published by Wiley Periodicals LLC This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/3.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Methods Nell, Rogier J. van Steenderen, Debby Menger, Nino V. Weitering, Thomas J. Versluis, Mieke van der Velden, Pieter A. Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR |
title | Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR |
title_full | Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR |
title_fullStr | Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR |
title_full_unstemmed | Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR |
title_short | Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR |
title_sort | quantification of dna methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital pcr |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756443/ https://www.ncbi.nlm.nih.gov/pubmed/32906203 http://dx.doi.org/10.1002/humu.24111 |
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