![]() Finally, there are limited validation data of microarray results by a gold-standard technique (bisulfite-PCR). The sensitivity of antibody-based methods is undetermined and possibly low (in our experience). Methods that rely on frequent sites (HpaII/MspI) result in a high genome fraction to amplify (high complexity), which limits PCR efficiency and ends up favoring non-CpG island DNA. There are major problems with each published method. 2004) or antibodies that recognize 5-methyl cytidine ( Weber et al. Most of the published methods aim at selective enrichment for the methylated fraction of the genome, using either methylation-sensitive restriction enzymes ( Yan et al. Several platforms are available, with variation in genome representation and probe size (oligonucleotides, short DNA fragments, or BACs).Ī major issue in methylation microarrays is the protocol for target preparation. ![]() Microarray chips containing promoter sequences filled this gap. Although efficient, both methods lack the high throughput required to study large sample collections for clinical and/or epidemiological purposes. 2000) and also revealed the existence of coordinated hypermethylation of multiple genes in subsets of samples, a process termed CpG island methylator phenotype ( Toyota et al. The use of techniques that test DNA methylation in an unbiased way, such as MCA (methylated CpG island amplification) and RLGS (restriction landmark genomic scanning), revealed that DNA methylation in cancer happens in a tissue-specific pattern ( Costello et al. Much of the knowledge about aberrant DNA methylation in cancer came from genome-wide investigations. While normal patterns of DNA methylation are important for genomic imprinting, X-chromosome inactivation, and to repress mobilization of repetitive elements, aberrant DNA methylation in cancer is associated with silencing of tumor-suppressor genes and genes involved in invasion, angiogenesis, and apoptosis ( Sugimura and Ushijima 2000 Toyota and Issa 2005). This happens by the enzymatic addition of methyl groups to CpG dinucleotides in an orchestrated reaction that involves DNA methyltransferases, methyl-binding domain proteins, and histone deacetylases ( Herman and Baylin 2003 Laird 2005). One of the main causes of epigenetic silencing in cancer is DNA methylation of cytosines in CG-rich regions (CpG islands) close to gene promoters. In cancer, loss of expression of selected genes happens by either genetic mutation or epigenetic silencing. In summary, MCAM is a suitable technique to discover methylated genes and to profile methylation changes in clinical samples in a high-throughput fashion. Unsupervised hierarchical clustering segregated the tumors into the expected subgroups based on CpG island methylator phenotype classification. The sensitivity and specificity of the method to detect hypermethylated loci were 88% and 96%, respectively, according to validation by bisulfite-PCR. We validated this method in three cancer cell lines and 15 primary colorectal tumors, resulting in the discovery of hundreds of new methylated genes in cancer. Here we overcome this limitation and present an improved method to identify methylated genes genome-wide by hybridizing a CpG island microarray with amplicons obtained by the methylated CpG island amplification technique (MCAM). The lack of high-throughput methods with high specificity and sensitivity to detect changes in DNA methylation has limited its application for clinical profiling. An abnormal pattern of DNA methylation occurs at specific genes in almost all neoplasms. ![]()
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