Rapid quantification of global DNA methylation by isocratic cation exchange high-performance liquid chromatography

Wilfried Rozhon; Tuncay Baubec; Juliane Mayerhofer; Ortrun Mittelsten Scheid; Claudia Jonak

doi:10.1016/j.ab.2008.01.001

Rapid quantification of global DNA methylation by isocratic cation exchange high-performance liquid chromatography

Wilfried Rozhon, Tuncay Baubec, Juliane Mayerhofer, Ortrun Mittelsten Scheid, Claudia Jonak

Gregor Mendel Institute of Molecular Plant Biology

Research output: Contribution to journal › Article › peer-review

67 Scopus citations

Abstract

The DNA of many eukaryotes is methylated at specific cytosine residues in connection with gene regulation. Here we report a method for the quantification of global cytosine methylation based on enzymatic hydrolysis of DNA, dephosphorylation, and subsequent high-performance cation exchange chromatography. Nucleosides are separated in less than 3 min under isocratic conditions on a benzenesulfonic acid-modified silica phase and detected by UV absorption. As little as 1 μg of DNA is sufficient to measure 5-methyldeoxycytosine levels with a typical relative standard deviation of less than 3%. As a proof of concept, the method was applied for analysis of DNA from several Arabidopsis thaliana mutants affected in DNA methylation and from Medicago sativa seedlings treated with the environmental pollutant chromium(VI).

Original language	English
Pages (from-to)	354-360
Number of pages	7
Journal	Analytical Biochemistry
Volume	375
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2008.01.001
State	Published - 15 Apr 2008
Externally published	Yes

Keywords

5-Methyl-2′-deoxycytidine
DNA methylation
Deoxycytidine
Epigenetics
High-performance cation exchange chromatography

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10.1016/j.ab.2008.01.001

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title = "Rapid quantification of global DNA methylation by isocratic cation exchange high-performance liquid chromatography",

abstract = "The DNA of many eukaryotes is methylated at specific cytosine residues in connection with gene regulation. Here we report a method for the quantification of global cytosine methylation based on enzymatic hydrolysis of DNA, dephosphorylation, and subsequent high-performance cation exchange chromatography. Nucleosides are separated in less than 3 min under isocratic conditions on a benzenesulfonic acid-modified silica phase and detected by UV absorption. As little as 1 μg of DNA is sufficient to measure 5-methyldeoxycytosine levels with a typical relative standard deviation of less than 3%. As a proof of concept, the method was applied for analysis of DNA from several Arabidopsis thaliana mutants affected in DNA methylation and from Medicago sativa seedlings treated with the environmental pollutant chromium(VI).",

keywords = "5-Methyl-2′-deoxycytidine, DNA methylation, Deoxycytidine, Epigenetics, High-performance cation exchange chromatography",

author = "Wilfried Rozhon and Tuncay Baubec and Juliane Mayerhofer and Scheid, {Ortrun Mittelsten} and Claudia Jonak",

note = "Funding Information: This work was supported by the Austrian Science Fund (P-16408-B12 and P-18986-B17), the Vienna Science and Technology Fund (FA546028), the EU NoE Epigenome (LSHG-CT-2004-503433), and the GEN-AU (GZ 200.140-VI/1/2006). We thank Olivier Mathieu and Jerzy Paszkowski for met 1-3 seeds, Andrea Foerster for callus material, and Bettina Dekrout for technical assistance.",

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doi = "10.1016/j.ab.2008.01.001",

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T1 - Rapid quantification of global DNA methylation by isocratic cation exchange high-performance liquid chromatography

AU - Rozhon, Wilfried

AU - Baubec, Tuncay

AU - Mayerhofer, Juliane

AU - Scheid, Ortrun Mittelsten

AU - Jonak, Claudia

N1 - Funding Information: This work was supported by the Austrian Science Fund (P-16408-B12 and P-18986-B17), the Vienna Science and Technology Fund (FA546028), the EU NoE Epigenome (LSHG-CT-2004-503433), and the GEN-AU (GZ 200.140-VI/1/2006). We thank Olivier Mathieu and Jerzy Paszkowski for met 1-3 seeds, Andrea Foerster for callus material, and Bettina Dekrout for technical assistance.

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N2 - The DNA of many eukaryotes is methylated at specific cytosine residues in connection with gene regulation. Here we report a method for the quantification of global cytosine methylation based on enzymatic hydrolysis of DNA, dephosphorylation, and subsequent high-performance cation exchange chromatography. Nucleosides are separated in less than 3 min under isocratic conditions on a benzenesulfonic acid-modified silica phase and detected by UV absorption. As little as 1 μg of DNA is sufficient to measure 5-methyldeoxycytosine levels with a typical relative standard deviation of less than 3%. As a proof of concept, the method was applied for analysis of DNA from several Arabidopsis thaliana mutants affected in DNA methylation and from Medicago sativa seedlings treated with the environmental pollutant chromium(VI).

AB - The DNA of many eukaryotes is methylated at specific cytosine residues in connection with gene regulation. Here we report a method for the quantification of global cytosine methylation based on enzymatic hydrolysis of DNA, dephosphorylation, and subsequent high-performance cation exchange chromatography. Nucleosides are separated in less than 3 min under isocratic conditions on a benzenesulfonic acid-modified silica phase and detected by UV absorption. As little as 1 μg of DNA is sufficient to measure 5-methyldeoxycytosine levels with a typical relative standard deviation of less than 3%. As a proof of concept, the method was applied for analysis of DNA from several Arabidopsis thaliana mutants affected in DNA methylation and from Medicago sativa seedlings treated with the environmental pollutant chromium(VI).

KW - 5-Methyl-2′-deoxycytidine

KW - DNA methylation

KW - Deoxycytidine

KW - Epigenetics

KW - High-performance cation exchange chromatography

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U2 - 10.1016/j.ab.2008.01.001

DO - 10.1016/j.ab.2008.01.001

M3 - Article

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AN - SCOPUS:40649084031

SN - 0003-2697

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SP - 354

EP - 360

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 2

ER -

Rapid quantification of global DNA methylation by isocratic cation exchange high-performance liquid chromatography

Abstract

Keywords

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