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 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.
Tost
Jörg
J
Centre National de Génotypage, Bâtiment G2, 2 Rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France.
Schatz
Philipp
P
Schuster
Matthias
M
Berlin
Kurt
K
Gut
Ivo Glynne
IG
D001483:Base Sequence; D018899:CpG Islands / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005169:Factor VIII / Q000235:genetics; D005838:Genotype; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D004273:DNA, Neoplasm; D007527:Isoenzymes; D005169:Factor VIII; D004247:DNA; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase