Analysis of gene-specific DNA methylation patterns by pyrosequencing technology.
As the sequence of the human genome is now nearly finished, genome research turns to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the pathogenesis of various diseases. The real-time luminometric detection of pyrophosphate release upon nucleotide incorporation in the Pyrosequencing technology is ideally suited for the simultaneous analysis and quantification of the methylation degree of several CpG positions in close proximity. We developed and improved this analysis to obtain reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 80 nt. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity, which may be used as DNA methylation markers.
Tost
Jörg
J
Department of Technology Development, Centre National de Génotypage, Evry, France.
Gut
Ivo Glynne
IG
D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D019175:DNA Methylation; D011756:Diphosphates / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D015894:Genome, Human / Q000235:genetics; D006680:HLA Antigens / Q000032:analysis / Q000235:genetics; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000378:metabolism; D013698:Templates, Genetic
D011756:Diphosphates; D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite