Abstract:
: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.
journal_name
Genome Resjournal_title
Genome researchauthors
Galm O,Rountree MR,Bachman KE,Jair KW,Baylin SB,Herman JGdoi
10.1101/gr.202501subject
Has Abstractpub_date
2002-01-01 00:00:00pages
153-7issue
1eissn
1088-9051issn
1549-5469journal_volume
12pub_type
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