Semiautomatic detection of DNA methylation at CpG islands.

DNA methylation of genes is increasingly recognized as one of the major mechanisms of gene inactivation that may contribute to the development of human cancer by silencing genes that suppress tumor growth (1). In recent years, methylation-specific PCR (MSP) of bisulfite-modified genomic DNA (gDNA) has been used as a standard method to detect DNA methylation changes at CpG islands (2). MSP is a PCR-based method that requires much less gDNA than the traditional Southern blot analysis method. However, the MSP assay has to be set up individually for each gene using methylated and unmethylated primer pairs, and the result of the assay is determined by visual interpretation of the MSP products after agarose gel electrophoresis. Consequently, there is no objective cutoff value for MSP, and the determination of methylation changes is not quantitative. Here, we describe the methylation-specific single base extension (MSBE) assay that could detect DNA methylation at one or multiple CpG islands simultaneously in a semiautomatic fashion. This method is easy to set up and is more quantitative than the traditional MSP assay. It is also easily expandable for large, genome-wide screening of promoter-methylation status of many genes by the use of multiple primers. As shown in Figure 1, DNA fragments were first amplified using nonmethylation-sensitive primers and sodium bisulfite-modified gDNA (2). After purification of the PCR product, a single base was extended at the predetermined CpG site with primers that complemented to either the methylated Semiautomatic detection of DNA methylation at CpG islands