Methylated CpG Island Recovery Assay Research Articles

MIRA-Assisted Microarray Analysis, a New Technology for the Determination of DNA Methylation Patterns, Identifies Frequent Methylation of Homeodomain-Containing Genes in Lung Cancer Cells

We present a straightforward and comprehensive approach for DNA methylation analysis in mammalian genomes. The methylated-CpG island recovery assay (MIRA), which is based on the high affinity of the MBD2/MBD3L1 complex for methylated DNA, has been used to detect cell type-dependent differences in DNA methylation on a microarray platform. The procedure has been verified and applied to identify a series of novel candidate lung tumor suppressor genes and potential DNA methylation markers that contain methylated CpG islands. One gene of particular interest was DLEC1, located at a commonly deleted area on chromosome 3p22-p21.3, which was frequently methylated in primary lung cancers and melanomas. Among the identified methylated genes, homeodomain-containing genes were unusually frequent (11 of the top 50 hits) and were targeted on different chromosomes. These genes included LHX2, LHX4, PAX7, HOXB13, LBX1, SIX2, HOXD3, DLX1, HOXD1, ONECUT2, and PAX9. The data show that MIRA-assisted microarray analysis has a low false-positive rate and has the capacity to catalogue methylated CpG islands on a genome-wide basis. The results support the hypothesis that cancer-associated DNA methylation events do not occur randomly throughout the genome but at least some are targeted by specific mechanisms.

Methylated-CpG island recovery assay: a new technique for the rapid detection of methylated-CpG islands in cancer

Hypermethylation of CpG islands is a phenomenon commonly observed during the development and progression of human tumors. Detection of methylated-CpG islands in easily accessible biological materials such as serum has the potential to be useful for the early diagnosis of cancer. Most currently used methods for detecting methylated-CpG islands are based on sodium bisulfite conversion of genomic DNA, followed by PCR reactions. Here we describe a method, methylated-CpG island recovery assay (MIRA) that does not depend on the use of sodium bisulfite but has similar sensitivity and specificity as bisulfite-based approaches. Methyl-CpG-binding domain proteins, such as methyl-CpG-binding domain protein-2 (MBD2), have the capacity to bind specifically to methylated DNA sequences. In the MIRA procedure, sonicated genomic DNA isolated from cells or tissue is incubated with a matrix containing glutathione-S-transferase-MBD2b in the presence of methyl-CpG-binding domain protein 3-like-1, a binding partner of MBD2 that increases the affinity of MBD2 for methylated DNA. Specifically bound DNA is eluted from the matrix and gene-specific PCR reactions are performed to detect CpG island methylation. Methylation can be detected using 1 ng of DNA or 3000 cells. MIRA is a specific and sensitive, but not laborious, technique that can be clinically useful in the detection and diagnosis of any DNA methylation-associated disease, including cancer.