Rapid and sensitive detection of CpG-methylation using methyl-binding (MB)-PCR

Abstract

Methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. We have developed a novel technique for detecting CpG-methylated DNA termed methyl-binding (MB)-PCR. This technique utilizes a recombinant protein with high affinity for CpG-methylated DNA that is coated onto the walls of a PCR vessel and selectively captures methylated DNA fragments from a mixture of genomic DNA. The retention and, hence, the degree of methylation of a specific DNA fragment (e.g. a CpG island promoter of a specific gene) is detected in the same tube by gene-specific PCR. MB-PCR does not require bisulfite treatment or methylation-sensitive restriction and provides a quick, simple and extremely sensitive technique allowing the detection of methylated DNA, in particular in tumor tissue or tumor cells from limited samples. Using this novel approach, we determined the methylation status of several established and candidate tumor suppressor genes and identified the ICSBP gene, encoding the myeloid and B-cell-specific transcription factor interferon consensus sequence-binding protein, as a target for aberrant hypermethylation in acute myeloid leukemia.

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes

“…Our eMethylsorb detection technique is also not limited to availability of methylation-sensitive restriction enzyme sites used in other methylation assays. 6,35,36 Moreover, the RSD over three independent eMethylsorb assays with single-use disposable SPE-Au is 5.3% (Fig. 2B), showing good assay reproducibility.…”

Wilms’ tumor gene 1 protein represses the expression of the tumor suppressor interferon regulatory factor 8 in human hematopoietic progenitors and in leukemic cells

“…[39][40][41] In AML, 7 out of 21 analyzed samples showed signs of promoter methylation. 42 However, high expression of IRF8 in the presence of methylated promoter has been reported for leukemic cell lines. 43 To investigate whether WT1-induced repression of IRF8 correlated with increased methylation of the IRF8 gene, we performed bisulfite sequencing of DNA from human progenitor cells transduced with WT1.…”

Flow Cytometric and Laser Scanning Microscopic Approaches in Epigenetics Research

“…Moreover, global changes of certain epigenetic modifications may have their independent diagnostic value, especially when analyzed in correlation with other phenotypic markers, an opportunity offered by up-to-date laser scanning microscopic systems (20,21). Development of antibodies and chimeric methyl CpG-binding antibody-like proteins (24)(25)(26)(27), both recognizing CpG with high specificity, has opened novel perspectives for the diagnostic analysis of global methylation states. Anti-5mC antibodies are commercially available through various sources (e.g., Abcam and Biocarta US).…”

“…In experiments using the recombinant mCpG-binding antibody-like MBD-Fc protein (26)(27)(28), the overall level of CpG methylation has been quantified in the HCT116 cell line (Fig.7.2). As shown by confocal laser scanning microscopy …”