Rapid and sensitive detection of CpG-methylation using methyl-binding (MB)-PCR.
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.
Gebhard
Claudia
C
Department of Hematology and Oncology, University Hospital, 93042, Regensburg, Germany.
Schwarzfischer
Lucia
L
Pham
Thu Hang
TH
Andreesen
Reinhard
R
Mackensen
Andreas
A
Rehli
Michael
M
D000208:Acute Disease; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000032:analysis* / Q000737:chemistry; D004268:DNA-Binding Proteins; D016147:Genes, Tumor Suppressor; D006801:Humans; D050835:Interferon Regulatory Factors / Q000235:genetics; D007951:Leukemia, Myeloid / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D013997:Time Factors; D014407:Tumor Cells, Cultured; D000099185:Interferon Regulatory Factor-8
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D050835:Interferon Regulatory Factors; D000099185:Interferon Regulatory Factor-8; C489325:MBD2 protein, human