Enzymatic regional methylation assay: a novel method to quantify regional CpG methylation density.
We have developed a novel quantitative method for rapidly assessing the CpG methylation density of a DNA region in mammalian cells. After bisulfite modification of genomic DNA, the region of interest is PCR amplified with primers containing two dam sites (GATC). The purified PCR products are then incubated with 14C-labeled S-adenosyl-L-methionine (SAM) and dam methyltransferase as an internal control to standardize DNA quantity. This is followed by an incubation with 3H-labeled SAM and SssI methyltransferase for methylation quantification. By use of standard mixtures of cell line DNA with a defined methylation status in every assay, the ratio (3H/14C signal) of each sample can be converted into percentage values to assess the methylation density of the amplified sequence. This methylation-sensitive technique, termed ERMA (Enzymatic Regional Methylation Assay) provides several advantages over existing methods used for methylation analysis as it determines an exact measurement of the methylation density of the region studied. We demonstrate a use of this technique in determining the methylation density of the promoter region of the tumor suppressor gene p15INK4B and changes that occur after treatment with demethylating agents.
Galm
Oliver
O
Oncology Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.
Rountree
Michael R
MR
Bachman
Kurtis E
KE
Jair
Kam-Wing
KW
Baylin
Stephen B
SB
Herman
James G
JG
D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry* / Q000235:genetics; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D016147:Genes, Tumor Suppressor; D018922:HL-60 Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D012436:S-Adenosylmethionine / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D025521:Tumor Suppressor Proteins; D012436:S-Adenosylmethionine; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)