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PMID: 12711695 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry.

Nucleic acids research ·Vol. 31 ·No. 9 ·2003-05-01 ·Pages e50

Tost J, Schatz P, Schuster M, Berlin K, Gut IG

Abstract

As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R(2) >or= 0.99 and standard deviation around 2% for the different positions. The limit of detection was approximately 5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.

MeSH Terms
Base Sequence CpG Islands/genetics DNA/chemistry,genetics,metabolism DNA Methylation DNA, Neoplasm/chemistry,genetics,metabolism Factor VIII/genetics Genotype Glutathione S-Transferase pi Glutathione Transferase/genetics Humans Isoenzymes/genetics Molecular Sequence Data Reproducibility of Results Sequence Analysis, DNA/methods Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
Chemicals
DNA, Neoplasm Isoenzymes Factor VIII DNA GSTP1 protein, human Glutathione S-Transferase pi Glutathione Transferase
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Tost Jörg
Centre National de Génotypage, Bâtiment G2, 2 Rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France.
Schatz Philipp
Schuster Matthias
Berlin Kurt
Gut Ivo Glynne
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gng050
Published
2003-05-01
Pages
e50
Language
English
Region
England
NLM ID
0411011
PMCID
PMC154238
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/gng050 PMC Full Text View journal details
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