Monitoring methylation changes in cancer.

Advances in biochemical engineering/biotechnology (2007-02-13)
Verena Beier, Cora Mund, Jörg D Hoheisel
ABSTRACT

Methylation of cytosines at their carbon-5 position plays an important role both during development and in tumorgenesis. The methylation occurs almost exclusively in CpG dinucleotides. While the bulk of human genomic DNA is depleted in CpG sites, there are CpG-rich stretches, so-called CpG islands, which are located in promoter regions of more than 70% of all known human genes. In normal cells, CpG islands are unmethylated, reflecting an transcriptionally active state of the respective gene. Epigenetic silencing of tumor suppressor genes by hypermethylation of CpG islands is a very early and stable characteristic of tumorigenesis. The detection of DNA methylation is based on a treatment of genomic DNA with sodium bisulfite, which converts only unmethylated cytosines to uracil, while methylated cytosines stay unaltered. This sequence conversion can be detected in the same way as a single nucleotide polymorphism. Even though different approaches have been established for analysing DNA methylation, so far detection methods that are capable of surveying the methylation status of multiple gene promoters have been restricted to a limited number of cytosines. The use of oligonucleotide microarrays permits the parallel analysis of the methylation status of individual cytosines on a genome-wide and gene-specific level. On the one hand, a hybridization-based setup is described employing microarrays that contain oligonucleotide probes of 17-25 bases in length reflecting the methylated as well as the unmethylated status of each CpG site. After hybridization of sodium bisulfite treated and fluorescently labeled targets, methylation status of individual CpG dinucleotides can be computed based on resulting signal intensities. Secondly, a microarray-based approach for detecting methylation-specific sequence polymorphisms via an on-chip enzymatic primer extension is described.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Imprint® Methylated DNA Quantification Kit, To measure global DNA methylation shifts from as low as 10 ng DNA
Sigma-Aldrich
Imprint® DNA Modification Kit, For bisulfite DNA conversion & purification