Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry

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...

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Bibliographic Details
Published in:Nucleic acids research 2003-05, Vol.31 (9), p.e50-e50
Main Authors: Tost, Jörg, Schatz, Philipp, Schuster, Matthias, Berlin, Kurt, Gut, Ivo Glynne
Format: Article
Language:English
Subjects:
Base Sequence
CpG Islands - genetics
DNA - chemistry
DNA - genetics
DNA - metabolism
DNA Methylation
DNA, Neoplasm - chemistry
DNA, Neoplasm - genetics
DNA, Neoplasm - metabolism
Factor VIII - genetics
Genotype
Glutathione S-Transferase pi
Glutathione Transferase - genetics
Humans
Isoenzymes - genetics
Molecular Sequence Data
NAR Methods Online
Reproducibility of Results
Sequence Analysis, DNA - methods
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
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Summary: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 R2 ≥ 0.99 and standard deviation around 2% for the different positions. The limit of detection was ∼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.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gng050